EXPERIENCE AND REASON |
Recombinant Granulocyte Colony-Stimulating Factor Administered Enterally to Neonates Is Not Absorbed
Division of Neonatology, Department of Pediatrics, University of South Florida College of Medicine, All Children’s Hospital, St Petersburg, FL 33710
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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Granulocyte colony-stimulating factor (G-CSF) is present in liquids swallowed by the fetus and neonate; specifically, amniotic fluid, colostrum, and human milk. The swallowed G-CSF has local effects on enteric cells, which express the G-CSF receptor. However, some portion of the G-CSF ingested by the fetus and neonate might be absorbed into the circulation and have systemic actions, such as stimulating neutrophil production. To assess this possibility we sought to determine if circulating G-CSF concentrations of neonates increase after enteral administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF). This was a single-center, prospective, blinded, randomized, 2 x 2 crossover study, with each infant receiving 1 dose of rhG-CSF (100 µg/kg) and 1 dose of placebo. Plasma G-CSF concentrations were measured at 2 and 4 hours after administration of the test solution. No significant change in plasma G-CSF concentration was observed after the enteral administration of rhG-CSF. On this basis, we conclude that orally administered rhG-CSF is not absorbed in significant quantities, and we speculate that the G-CSF swallowed by the fetus and neonate has local but not systemic effects.
Key Words: intestine • cytokines • development • neonates
Abbreviations: G-CSF-R, receptor for granulocyte colony-stimulating factor • G-CSF, granulocyte colony-stimulating factor • rhG-CSF, recombinant human granulocyte colony-stimulating factor • SD, standard deviation • IGF, insulin-like growth factor
"In Medicine one must pay attention not to plausible theorizing but to experience and reason together....I agree that theorizing is to be approved, provided that it is based on facts, and systematically makes its deductions from what is observed....Butconclusions drawn from unaided reason can hardly be serviceable; only those drawn from observed fact." Hippocrates: Precepts. (Short communications of factual material are published here. Comments and criticisms appear as letters to the Editor.)
Enterocytes in the developing human bowel express, on their luminal surface, specific receptors for granulocyte colony-stimulating factor (G-CSF-R).1,2 These receptors are frequently in proximity to their cognate ligand, granulocyte colony-stimulating factor (G-CSF), because G-CSF is present in relatively high concentrations in the liquids swallowed by the fetus and neonate; namely, amniotic fluid, colostrum, and human milk.2,3 G-CSF swallowed by the fetus and neonate appears to be relatively protected from degradation in the stomach and small intestine.4 Thus, the G-CSF swallowed by the fetus and neonate likely has either local actions in the gastrointestinal tract, or has systemic actions, or perhaps it has both. Systemic actions are likely only if the swallowed G-CSF is absorbed in a functional state and is subsequently transported through the circulation to systemic sites of actions, such as the bone marrow. The specific aim of this study was to determine if it is likely that the G-CSF ingested by the human fetus and neonate has systemic actions. To determine this, we enterally administered recombinant human granulocyte colony-stimulating factor (rhG-CSF) or placebo to neonates in a randomized, crossover trial, and quantified subsequent plasma G-CSF concentrations.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The investigation was a blinded, randomized, 2 x 2 crossover study of plasma G-CSF concentrations in preterm and term infants <1 month old. Subjects were randomized to 1 of 2 feeding sequences; either 100 µg/kg of rhG-CSF then placebo, or placebo then 100 µg/kg of rhG-CSF, with 48 hours between the 2 feedings. G-CSF concentrations were determined in human milk before the addition of the study drug, rhG-CSF, or placebo to the milk. Five percent dextrose water was used as the placebo, and was given in the exact same volume as that of the rhG-CSF. The quantity of rhG-CSF administered enterally was 10 times the intravenous or subcutaneous dose given to neonates for the treatment of neutropenia (10 µg/kg).
Whole blood (400 µL, ethylenediaminetetraacetic acid anticoagulant) was obtained for G-CSF concentration at 2 and 4 hours after the feeding. Plasma was collected and stored at –80°C until time of assay. We based the timing of plasma G-CSF measurements on our previous study, in which erythropoietin concentrations were obtained at 2, 4, 6, 12, and 24 hours after enteral erythropoietin dosing, and only the 2-hour samples were slightly elevated.5
Patient randomization was prospectively blocked into 2 cells, depending on gestational age at the time of study entry (
35 complete weeks of gestation, and >35 weeks of gestation). The primary outcome measure was plasma G-CSF concentration 2 and 4 hours after the oral dosing. Using our pilot data, we found that the plasma G-CSF concentrations in normal, nonneutropenic neonates varied with a standard deviation (SD) of 25 pg/mL. Assuming that a difference of 1 SD would be clinically/biologically significant, a sample size of 17 neonates (for a repeated measures study format) was estimated to be sufficient for 80% statistical power at an 
= 0.05.
Neonates admitted to the neonatal intensive care unit of Shands Children’s Hospital at the University of Florida were considered eligible for study if they were <1 month old at time of study entry, if they were deemed not to have an infection by the attending neonatologist at study entry, and were receiving at least 100 mL/kg/d enteral feedings of human milk. All subjects were randomized to treatment sequence using randomization blocked on whether the infant was preterm or term. This ensured a balanced randomization of treatment sequence within the preterm and term groups. The Institutional Review Board of the University of Florida approved the study, and the parents or guardians of each study subject signed informed consent documents.
Human milk and neonatal plasma G-CSF concentrations were determined by enzyme-linked immunosorbent assay (R&D System, Minneapolis, MN). A standard curve was done in duplicate using control solutions ranging from 0 to 2500 pg/mL. Intra-assay variability was <3% and sensitivity was <20 pg/mL. This assay has been tested for cross-reactivity with other cytokines, and no significant cross-reactivity or interference has been observed. Previously, we standardized the enzyme-linked immunosorbent assay for measurement of G-CSF in human milk.2
Factors to be included in the model were treatment (rhG-CSF vs placebo), term status (preterm vs term), and blood draw time (2 hours vs 4 hours). Specific contrasts at the following time points were of interest: 2-hour rhG-CSF versus 2-hour placebo and 4-hour rhG-CSF versus 4-hour placebo. The data were analyzed with multiple factor, repeated measures analysis of variance. The within-subjects variables included treatment (rhG-CSF vs placebo), and blood draw time (2 hours, and 4 hours), and comparisons were also made along the gestational age groups. Single variable comparisons were made with Student t tests (paired and unpaired, as applicable).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Twenty patients completed the study. Fourteen infants were
35 weeks’ gestation and 6 were >35 weeks’ gestation. All patients tolerated the enteral rhG-CSF well, with no apparent change in feeding tolerance or in stooling pattern. The weight (mean ± SD) of infants 35 weeks’ gestation at study entry was 1462 ± 367 g, while that of the infants >35 weeks’ gestation was 3850 ± 476 g. The mean day of life that infants <35 weeks’ gestation began the study was 18.3 ± 12.0 days (range: 4–31 days), while for infants >35 weeks’ gestation, the average day of life for study entry was 27.3 ± 18.2 days (range: 13–51 days). The concentration of G-CSF in human milk before the addition of rhG-CSF was 34 ± 19 pg/mL in the milk of mothers whose infants were 35 weeks’ gestation and was 29 ± 14 pg/mL in the milk of mothers whose infants were >35 weeks’ gestation (P = .53; preterm vs term). Similarly, there was no difference in the endogenous concentration of G-CSF in human milk samples on day 1 of the study when compared with day 4 of the study (P = .807).
Figure 1 shows the distribution of G-CSF concentrations in all subjects based on the treatment group. We performed a repeated measures analysis of variance with the within-subjects variables being treatment (rhG-CSF vs placebo) and blood draw time (2 hours vs 4 hours) with comparisons made along the gestational age groups. No effect of rhG-CSF administration on plasma G-CSF concentration was noted when treatment versus placebo group was compared regardless of gestational age (P = .686) or blood draw time (2 hours vs 4 hours; P = .806). When infants were blocked by gestational age, mean G-CSF concentrations at 2 and 4 hours after rhG-CSF dosing were no different in infants 35 weeks’ gestation than in those >35 weeks’ gestation (P = .503), and there was no difference between gestational age groups related to the blood draw time (P = .643). The outlier represented by an asterisk corresponds to the same patient, whereas the outliers represented by the circles are 2 different patients.
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) show the range of the values, which fall within 1.5 box-length spreads of the hinges. Values >1.5 box-lengths away from the box are plotted as o(outliers), and values 3.0 box-lengths away from the box are plotted as *(extremes), respectively. |
DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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A protective effect of human milk feedings, against bacterial infections in neonates, has been reported from the United States, Sweden, Guatemala, India, Bangladesh, Australia, and Great Britain.2 Several macromolecules are absorbed intact into the circulation of preterm infants, suggesting they have a systemic role.6 Some of the immunologic factors passively acquired by human milk feeding have been identified. These include immunoglobulins, complement components, interferon, cytokines, fatty acids, gangliosides, polysaccharides, glycoproteins, lymphocyte-derived chemotactic and migration inhibition factors, macrophages, granulocytes, lymphocytes, and epithelial cells.6
G-CSF is an 18.8-kDa glycoprotein that participates in the regulation of neutrophil production.7,8 It does this after binding with its specific receptor by inhibiting apoptosis of granulocytic progenitors and by supporting their clonal proliferation and differentiation. In addition to its presence on neutrophils and their progenitors, functional G-CSF-Rs are expressed on a variety of nonhematopoietic cells, including enterocytes of the fetus and neonate.1,2 In utero, fetal enterocytes are universally exposed to intraluminal G-CSF, which is present in amniotic fluid and continually swallowed by the fetus.3 Postnatally, infants fed human milk are also exposed to enteric G-CSF, as human milk contains significant concentrations of G-CSF.2,4
Local effects of G-CSF in the developing intestine have been established,9 but the focus of this study was to determine if enterally administered rhG-CSF is absorbed, and might therefore also have systemic effects such as on neutrophil production. We did not find an increase in plasma G-CSF concentration either 2 or 4 hours after the administration of a large enteral G-CSF dose. The dose of rhG-CSF we administered (100 µg/kg) was 100 times the normal daily fetal enteral intake of G-CSF consumed by swallowing amniotic fluid.10 We reasoned that if no G-CSF was detected in the plasma of neonates after swallowing such a pharmacologic dose, absorption was unlikely to occur after swallowing physiologic quantities. We conclude that rhG-CSF administered orally to neonates is absorbed very poorly if at all, and acknowledge that our selection of sample times could have influenced our results. On this basis, we speculate that the G-CSF swallowed by the fetus and neonate is not absorbed, but has a local action in the developing gastrointestinal tract. Studies related to insulin-like growth factor (IGF) have shown that human milk-borne IGF and IGF in amniotic fluid can enter the portal circulation intact and might have effects on the liver.11,12
This study addressed the enteral absorption of rhG-CSF in preterm and term neonates who were an average of 2 weeks old at study entry. Developmental changes in the ability of the small intestinal epithelium to take up and transfer into blood various macromolecules has been described in different animal species.9,13,14 In a study of term human neonates, Catassi and colleagues15 reported that the intestinal permeability of sugar is developmentally regulated and is different in infants fed human milk compared with those who are fed infant formula. When considering the variables of age and type of feeding, we cannot exclude the possibility that enterally administered rhG-CSF might be absorbed in neonates <1 week old or in those infants who are formula-fed.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants HD-01180 and HD-42326 from the National Institutes of Health.
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FOOTNOTES |
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Received for publication Jul 23, 2002; Accepted Feb 5, 2003.
Reprint requests to (D.A.C.) Division of Neonatology, Department of Pediatrics, University of South Florida, All Children’s Hospital, Children’s Research Institute-CRI 2006, 140 Seventh Ave South, St Petersburg, FL 33710. E-mail: dcalhoun{at}hsc.usf.edu
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REFERENCES |
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PEDIATRICS (ISSN 1098-4275). ©2003 by the American Academy of Pediatrics
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