Eur J Pediatr (1991) 150 : 767-772 034061999100160W
European Journal of
Pediatrics
9 Springer-Verlag199i
Weekly intravenous administration of recombinant human erythropoietin in infants with the anaemia of prematurity D. Beck 1, E. Masserey 1, M. Meyer 2, and A. Calame 1 ~Department of Paediatrics, 2Haemato-oncotogy Research Laboratory, Department of Paediatrics, University Hospital, CH-1011 Lausanne, Switzerland Received September 6, 1990 / Accepted February 27, 1991
Abstract. To study the safety and efficacy of administering human recombinant erythropoietin (rHuEPO) to infants with anaemia of prematurity, a combined phase I/ II trial of weekly intravenous injections for 4 weeks was undertaken. We treated 16 infants with 10, 25, 50, 100 or 200 units/kg body weight in groups of two to four patients per dose level. They were all born prematurely (mean gestational age: 29 weeks; range 27-32), had a mean post-natal age of 42 days (range: 25-59) and haemoglobin concentration of 87g/1 (range: 72-94) when treatment was started. Four patients (25%) needed a transfusion during the trial, one at day 7 treated with 10 units/ kg and 3 at days 15, 25, 29 with 100 units/kg. In the others, a progressive rise in mean haemoglobin values was seen in each group after 21 days of treatment, without a dosedependent effect. A positive change in absolute reticulocyte counts with a peak after 7-14 days of therapy was observed with low (25-50 units/kg) but not with higher doses, with a significant difference at day 14 between 25 and 100 units/kg (P < 0.01). A dose-limiting severe neutropenia (absolute neutrophil count < 0.5 • 109/1) occurred transiently in five patients, with doses > 25 units/kg. No infectious complication and no sign of iron deficiency were observed. Weekly low doses of rHuEPO appear safe, convenient to administer and able to induce a reticulocytic response in infants with anaemia of prematurity. A phase III placebo-controlled trial is needed to confirm these results. Neutropenia associated with rHuEPO administration in infants might be related to their stage of human ontogeny. Key words: Anaemia of prematurity - Recombinant erythropoietin - Neutropenia
haemoglobin concentration seen in all infants, with a nadir between 4 and 12 weeks of age and spontaneous recovery [2, 29]. In babies born prematurely, haemoglobin levels lower than 70 g/1 are frequently detected. More than 50% of those born after less than 32 weeks of gestation demonstrate inadequate adaptative responses with development of tachycardia, tachypnoea, feeding problems, diminished activity and possibly apnoeic attacks [2, 9, 29]. A blood transfusion is generally recommended for the treatment of symptomatic infants but controversies remain as to when to transfuse [21, 29]. The best predictor for transfusion could be gestational age < 30 w e e k s [5]. Although immunoreactive plasma erythropoietin (EPO) levels are sometimes increased at birth in premature babies, they fall rapidly after birth to concentrations lower than those of adults [4, 10, 31]. The mechanism of this EPO deficiency is not fully understood but is thought to be secondary to the immaturity of the hepatic sensor regulating EPO production by liver cells during fetal life [24]. Circulating [25] erythroid progenitor cells (burstforming unit, erythroid [BFU-E]), as well as those populating the marrow [19] can be detected during the anaemia of prematurity in higher numbers than found in normal adults; and BFU-E [25] appear to have in vitro a normal intrinsic responsiveness to naturally occuring human EPO or to the recombinant human hormone (rHuEPO). Since rHuEPO is now available for clinical use and therapeutic trials in anaemia of prematurity have been suggested [2, 30], we undertook a combined phase I/II study, as designed in one of the first trials in patients with the anaemia of end-stage renal disease [11].
Introduction
Patients and methods
Anaemia of prematurity is a well described clinical entity representing an exacerbation of the post-natal fall in
Ethical considerations
Offprint requests to: D. Beck Abbreviations: BFU-E = burst-forming unit, erythroid; EPO =
erythropoietin; rHuEPO = recombinant human erythropoietin
The study protocol was reviewed and approved by the ethics committee of the University of Lausanne medical school. Parental written informed consent was obtained for all infants enrolled in the study. In this first trial in infants, it was felt ethically better to treat patients when they were developing anaemia of prematurity rather than at a younger age in an attempt to prevent the anaemia from developing. The intravenous route of administration was
768 chosen because it was the only one extensively tested in humans when we designed our study [11, 32]. For the convenience of patients, who were clinically stable and without permanent intravenous line, we have used a single injection of r H u E P O each week.
Patients Premature infants were eligible for the trial only if they met all the following criteria: (1) gestational age at birth of no more than 32 weeks; (2) post-natal age of at least 28 days but no more than 12 weeks; (3) clinically stable condition including normal blood presure, normal paO2 while breathing room air, a previous pattern of weight gain while fed either by gavage or by mouth, no severe infection; (4) absence of a severe congenital malformation, hepatic or renal failure, or a haemostatic defect; (5) at least 2 haemoglobin determinations below 100 g/1. Three patients were excluded because their parents refused to sign informed consent. Gestational age at the time of birth was determined on the basis of maternal menstrual dates and was confirmed by neonatal examination.
Study design The combined phase I and II clinical trial was an open-label study of safety and efficacy with each dosage groups containing two to four patients. The patients received weekly intravenous injections of rHuEPO for 4 weeks or less if they received a blood transfusion, which was an endpoint of the study. Those completing the trial were observed for a period of 56 days from the beginning of treatment and the variables used for evaluation of response by groups were the changes in mean haemoglobin levels and absolute reticulocyte counts at weekly time intervals.
Recombinant human erythropoietin The hormone used in this study was produced by recombinant D N A technique and supplied by Cilag A.G. (Schaffausen, Switzerland). It is commercially available in Switzerland under the trade name E P R E X , with a specific activity of 119.000 units/mg of hormone according to informations provided by the manufacturer.
Clinical surveillance and laboratory tests All patients were hospitalized and under close supervision in our neonatal intermediate care unit at the time of enrollment and during the treatment period. Before the first injection of rHuEPO, baseqine studies included anthropometric measurements, physical examination, blood pressure determinations by the Doppler technique in the left arm; a complete blood count as determined by electronic blood cell counter, absolute neutrophil count calculated from 100-cell leucocyte differential count, reticulocyte count, liver function studies, serum iron, transferrin [3], ferritin and E P O concentration [20]. B F U - E were cultured as described by Shannon et al. [25] and counted according to the criteria of Gregory and Eaves [14]. Physical examination, weight, blood pressure, pulse determinations were performed daily during the administration of rHuEPO and the laboratory parameters were monitored weekly to assess any possible toxic effects. For comparative analysis of absolute neutrophil count, we retrospectively studied 25 untreated infants who were matched with the treated group for gestational, post-natal ages and sex. All were hospitalized during the same calendar year in our neonatal unit (the three excluded were among them) and blood counts were performed at the same post-natal age by both capillary and venous samplings, as for the study patients.
Table 1. Patient details before and during r H u E P O treatment Patient
At birth
At enrollment
(n)
GestaWeight tional age (g) (weeks)
Postnatal age (days)
Weight (g)
Haemoglobin level (g/l)
BFU-E Serum EPO (/105 cells) a levels (U/ml)
rHuEPO weekly doses (U/kg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
31 30 32 28 32 32 28 29 28 27 27 29 31 30 28 27
1450 660 1770 1000 1145 1270 1190 1120 1100 930 990 1170 1170 870 700 1140
25 37 42 59 39 51 38 50 44 46 40 31 35 47 48 42
1820 1000 2410 1400 2040 2060 1800 2280 1925 1430 1610 1550 1620 1810 1270 1880
92 89 87 87 86 90 82 90 72 94 89 84 87 80 86 91
36 • 260 • 187 • 75 • 62 • 254 • 137 • 69 • 125 • ND 82 • 102 • 189 • 40 • 308 • 249 •
10 10 25 25 25 50 50 50 100 100 100 100 200 200 200 200
Mean -- SD
29 _+2
1104 _+268
42 + 8
1744 + 370
87 + 5
a Mean _+ SD of quadruplicate tests b Untransfused patients only ND, Not done; NA, Not applicable
Treatment and evolution
24 60 56 34 24 80 48 20 5 12 35 24 4 44 80
5.7 10.7 6.8 13.2 6.9 ND 26.7 0.6 9.3 9.0 5.2 6.1 9.1 15.7 25.9 13.3 10.9 + 7.2
Transfusion (day of treatment)
+ (7)
+ (25) + (15) +(29)
Weight gain after 4 weeks b (g) 690 NA 1075 745 1060 820 740 660 815 NA NA NA 580 650 750 820 783 + 151
769 12(>
Iron and vitamin supply The routine administration of iron and vitamins for all prematurely born infants in our neonatal care unit was not modified for the study and consisted of iron sulphate 3 mg/kg daily per os starting at 6 weeks of age, alpha-tocophero120mg/kg per day intramuscularly during the first 3 days of life [28], followed by 5 mg/kg daily per os as part of a multivitamin preparation.
10@
--
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a51
Statistics
801
Student's t test was used for comparison of means of samples and the chi-square test with Yates correction for comparison of proportions. A P value of < 0.05 was considered as statistically significant,
75
2s
Results
0
2OO.
x v
Sixteen patients fulfilled the admission criteria and were treated according to the study protocol. Detailed patient data at birth and at the time of enrollment into the study are listed in Table 1. Serum EPO levels and circulating BFU-E numbers were within the age-related range of reported values [10, 25].
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Treatment and response The following weekly dose by single administration were given: 10 units/kg body weight (2 infants), 25 (3), 50 (3), 100 (4) and 200 (4). Symptomatic anaemia requiring transfusion developed in 4 (25%), including 1 and 3 patients treated with 10 and 100 units/kg respectively (Table 1). The increments in weight corresponded to expected values for age and were not influenced by doses of r H u E P O (Table 1). In the non-transfused and in the transfused infants before transfusion, the mean (+ SEM) haemoglobin values rose progressively during the trial without evidence of a dose-dependent effect (Fig. 1A). The mean reticulocyte counts increased transiently at dose levels of 25, 50, 100 units/kg, while no increment was seen at 10 or 200 units/kg (Fig. 1B). When plotting the mean differences from baseline values after 7, 14 and 21 days of treatment (Fig. 2), a bellshaped curve was obtained, with maximal values at doses of 25 (day 14) or 50 units/kg (day 7) followed by a trend toward negative results with further dose increments, reaching significance at day 14 between 25 and 100 units/kg (P < 0.01).
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European Journal of
Pediatrics
9 Springer-Verlag199i
Weekly intravenous administration of recombinant human erythropoietin in infants with the anaemia of prematurity D. Beck 1, E. Masserey 1, M. Meyer 2, and A. Calame 1 ~Department of Paediatrics, 2Haemato-oncotogy Research Laboratory, Department of Paediatrics, University Hospital, CH-1011 Lausanne, Switzerland Received September 6, 1990 / Accepted February 27, 1991
Abstract. To study the safety and efficacy of administering human recombinant erythropoietin (rHuEPO) to infants with anaemia of prematurity, a combined phase I/ II trial of weekly intravenous injections for 4 weeks was undertaken. We treated 16 infants with 10, 25, 50, 100 or 200 units/kg body weight in groups of two to four patients per dose level. They were all born prematurely (mean gestational age: 29 weeks; range 27-32), had a mean post-natal age of 42 days (range: 25-59) and haemoglobin concentration of 87g/1 (range: 72-94) when treatment was started. Four patients (25%) needed a transfusion during the trial, one at day 7 treated with 10 units/ kg and 3 at days 15, 25, 29 with 100 units/kg. In the others, a progressive rise in mean haemoglobin values was seen in each group after 21 days of treatment, without a dosedependent effect. A positive change in absolute reticulocyte counts with a peak after 7-14 days of therapy was observed with low (25-50 units/kg) but not with higher doses, with a significant difference at day 14 between 25 and 100 units/kg (P < 0.01). A dose-limiting severe neutropenia (absolute neutrophil count < 0.5 • 109/1) occurred transiently in five patients, with doses > 25 units/kg. No infectious complication and no sign of iron deficiency were observed. Weekly low doses of rHuEPO appear safe, convenient to administer and able to induce a reticulocytic response in infants with anaemia of prematurity. A phase III placebo-controlled trial is needed to confirm these results. Neutropenia associated with rHuEPO administration in infants might be related to their stage of human ontogeny. Key words: Anaemia of prematurity - Recombinant erythropoietin - Neutropenia
haemoglobin concentration seen in all infants, with a nadir between 4 and 12 weeks of age and spontaneous recovery [2, 29]. In babies born prematurely, haemoglobin levels lower than 70 g/1 are frequently detected. More than 50% of those born after less than 32 weeks of gestation demonstrate inadequate adaptative responses with development of tachycardia, tachypnoea, feeding problems, diminished activity and possibly apnoeic attacks [2, 9, 29]. A blood transfusion is generally recommended for the treatment of symptomatic infants but controversies remain as to when to transfuse [21, 29]. The best predictor for transfusion could be gestational age < 30 w e e k s [5]. Although immunoreactive plasma erythropoietin (EPO) levels are sometimes increased at birth in premature babies, they fall rapidly after birth to concentrations lower than those of adults [4, 10, 31]. The mechanism of this EPO deficiency is not fully understood but is thought to be secondary to the immaturity of the hepatic sensor regulating EPO production by liver cells during fetal life [24]. Circulating [25] erythroid progenitor cells (burstforming unit, erythroid [BFU-E]), as well as those populating the marrow [19] can be detected during the anaemia of prematurity in higher numbers than found in normal adults; and BFU-E [25] appear to have in vitro a normal intrinsic responsiveness to naturally occuring human EPO or to the recombinant human hormone (rHuEPO). Since rHuEPO is now available for clinical use and therapeutic trials in anaemia of prematurity have been suggested [2, 30], we undertook a combined phase I/II study, as designed in one of the first trials in patients with the anaemia of end-stage renal disease [11].
Introduction
Patients and methods
Anaemia of prematurity is a well described clinical entity representing an exacerbation of the post-natal fall in
Ethical considerations
Offprint requests to: D. Beck Abbreviations: BFU-E = burst-forming unit, erythroid; EPO =
erythropoietin; rHuEPO = recombinant human erythropoietin
The study protocol was reviewed and approved by the ethics committee of the University of Lausanne medical school. Parental written informed consent was obtained for all infants enrolled in the study. In this first trial in infants, it was felt ethically better to treat patients when they were developing anaemia of prematurity rather than at a younger age in an attempt to prevent the anaemia from developing. The intravenous route of administration was
768 chosen because it was the only one extensively tested in humans when we designed our study [11, 32]. For the convenience of patients, who were clinically stable and without permanent intravenous line, we have used a single injection of r H u E P O each week.
Patients Premature infants were eligible for the trial only if they met all the following criteria: (1) gestational age at birth of no more than 32 weeks; (2) post-natal age of at least 28 days but no more than 12 weeks; (3) clinically stable condition including normal blood presure, normal paO2 while breathing room air, a previous pattern of weight gain while fed either by gavage or by mouth, no severe infection; (4) absence of a severe congenital malformation, hepatic or renal failure, or a haemostatic defect; (5) at least 2 haemoglobin determinations below 100 g/1. Three patients were excluded because their parents refused to sign informed consent. Gestational age at the time of birth was determined on the basis of maternal menstrual dates and was confirmed by neonatal examination.
Study design The combined phase I and II clinical trial was an open-label study of safety and efficacy with each dosage groups containing two to four patients. The patients received weekly intravenous injections of rHuEPO for 4 weeks or less if they received a blood transfusion, which was an endpoint of the study. Those completing the trial were observed for a period of 56 days from the beginning of treatment and the variables used for evaluation of response by groups were the changes in mean haemoglobin levels and absolute reticulocyte counts at weekly time intervals.
Recombinant human erythropoietin The hormone used in this study was produced by recombinant D N A technique and supplied by Cilag A.G. (Schaffausen, Switzerland). It is commercially available in Switzerland under the trade name E P R E X , with a specific activity of 119.000 units/mg of hormone according to informations provided by the manufacturer.
Clinical surveillance and laboratory tests All patients were hospitalized and under close supervision in our neonatal intermediate care unit at the time of enrollment and during the treatment period. Before the first injection of rHuEPO, baseqine studies included anthropometric measurements, physical examination, blood pressure determinations by the Doppler technique in the left arm; a complete blood count as determined by electronic blood cell counter, absolute neutrophil count calculated from 100-cell leucocyte differential count, reticulocyte count, liver function studies, serum iron, transferrin [3], ferritin and E P O concentration [20]. B F U - E were cultured as described by Shannon et al. [25] and counted according to the criteria of Gregory and Eaves [14]. Physical examination, weight, blood pressure, pulse determinations were performed daily during the administration of rHuEPO and the laboratory parameters were monitored weekly to assess any possible toxic effects. For comparative analysis of absolute neutrophil count, we retrospectively studied 25 untreated infants who were matched with the treated group for gestational, post-natal ages and sex. All were hospitalized during the same calendar year in our neonatal unit (the three excluded were among them) and blood counts were performed at the same post-natal age by both capillary and venous samplings, as for the study patients.
Table 1. Patient details before and during r H u E P O treatment Patient
At birth
At enrollment
(n)
GestaWeight tional age (g) (weeks)
Postnatal age (days)
Weight (g)
Haemoglobin level (g/l)
BFU-E Serum EPO (/105 cells) a levels (U/ml)
rHuEPO weekly doses (U/kg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
31 30 32 28 32 32 28 29 28 27 27 29 31 30 28 27
1450 660 1770 1000 1145 1270 1190 1120 1100 930 990 1170 1170 870 700 1140
25 37 42 59 39 51 38 50 44 46 40 31 35 47 48 42
1820 1000 2410 1400 2040 2060 1800 2280 1925 1430 1610 1550 1620 1810 1270 1880
92 89 87 87 86 90 82 90 72 94 89 84 87 80 86 91
36 • 260 • 187 • 75 • 62 • 254 • 137 • 69 • 125 • ND 82 • 102 • 189 • 40 • 308 • 249 •
10 10 25 25 25 50 50 50 100 100 100 100 200 200 200 200
Mean -- SD
29 _+2
1104 _+268
42 + 8
1744 + 370
87 + 5
a Mean _+ SD of quadruplicate tests b Untransfused patients only ND, Not done; NA, Not applicable
Treatment and evolution
24 60 56 34 24 80 48 20 5 12 35 24 4 44 80
5.7 10.7 6.8 13.2 6.9 ND 26.7 0.6 9.3 9.0 5.2 6.1 9.1 15.7 25.9 13.3 10.9 + 7.2
Transfusion (day of treatment)
+ (7)
+ (25) + (15) +(29)
Weight gain after 4 weeks b (g) 690 NA 1075 745 1060 820 740 660 815 NA NA NA 580 650 750 820 783 + 151
769 12(>
Iron and vitamin supply The routine administration of iron and vitamins for all prematurely born infants in our neonatal care unit was not modified for the study and consisted of iron sulphate 3 mg/kg daily per os starting at 6 weeks of age, alpha-tocophero120mg/kg per day intramuscularly during the first 3 days of life [28], followed by 5 mg/kg daily per os as part of a multivitamin preparation.
10@
--
9sl 901
a51
Statistics
801
Student's t test was used for comparison of means of samples and the chi-square test with Yates correction for comparison of proportions. A P value of < 0.05 was considered as statistically significant,
75
2s
Results
0
2OO.
x v
Sixteen patients fulfilled the admission criteria and were treated according to the study protocol. Detailed patient data at birth and at the time of enrollment into the study are listed in Table 1. Serum EPO levels and circulating BFU-E numbers were within the age-related range of reported values [10, 25].
I
175.
15(>,
rl
125" o
I
10O. a~
g:
75 50
Treatment and response The following weekly dose by single administration were given: 10 units/kg body weight (2 infants), 25 (3), 50 (3), 100 (4) and 200 (4). Symptomatic anaemia requiring transfusion developed in 4 (25%), including 1 and 3 patients treated with 10 and 100 units/kg respectively (Table 1). The increments in weight corresponded to expected values for age and were not influenced by doses of r H u E P O (Table 1). In the non-transfused and in the transfused infants before transfusion, the mean (+ SEM) haemoglobin values rose progressively during the trial without evidence of a dose-dependent effect (Fig. 1A). The mean reticulocyte counts increased transiently at dose levels of 25, 50, 100 units/kg, while no increment was seen at 10 or 200 units/kg (Fig. 1B). When plotting the mean differences from baseline values after 7, 14 and 21 days of treatment (Fig. 2), a bellshaped curve was obtained, with maximal values at doses of 25 (day 14) or 50 units/kg (day 7) followed by a trend toward negative results with further dose increments, reaching significance at day 14 between 25 and 100 units/kg (P < 0.01).
B
225.
251
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