Ronald J. McPherson

A Phase I/II Trial of High-Dose Erythropoietin in Extremely Low Birth Weight Infants: Pharmacokinetics and Safety

OBJECTIVES. High-dose recombinant erythropoietin is neuroprotective in animal models of neonatal brain injury. Extremely low birth weight infants are at high risk for brain injury and neurodevelopmental problems and might benefit from recombinant erythropoietin. We designed a phase I/II trial to test the safety and determine the pharmacokinetics of high-dose recombinant erythropoietin in extremely low birth weight infants. METHODS. In a prospective, dose-escalation, open-label trial, we compared 30 infants who were treated with high-dose recombinant erythropoietin with 30 concurrent control subjects. Eligible infants were <24 hours old, ≤1000 g birth weight, and ≤28 weeks of gestation and had an umbilical artery catheter in place. Each infant received 3 intravenous doses of 500, 1000, or 2500 U/kg at 24-hour intervals beginning on day 1 of age. Blood samples were collected at scheduled intervals to determine recombinant erythropoietin pharmacokinetics. Safety parameters were also evaluated. In the concurrent control group, only clinical data were collected. RESULTS. Mean erythropoietin concentrations 30 minutes after recombinant erythropoietin infusion were 5973 ± 266, 12291 ± 403, and 34197 ± 1641 mU/mL after 500, 1000, or 2500 U/kg, respectively. High-dose recombinant erythropoietin followed nonlinear pharmacokinetics as a result of decreasing clearance from the lowest dosage (17.3 mL/hour per kg for 500 U/kg) to the highest dosage (8.2 mL/hour per kg for 2500 U/kg). Steady state was achieved within 24 to 48 hours. Both 1000 and 2500 U/kg recombinant erythropoietin produced peak serum erythropoietin concentrations that were comparable to neuroprotective concentrations that previously were seen in experimental animals. No excess adverse events occurred in the recombinant erythropoietin–treated infants compared with control infants. CONCLUSIONS. Early high-dose recombinant erythropoietin is well tolerated by extremely low birth weight infants, causing no excess morbidity or mortality. Recombinant erythropoietin dosages of 1000 and 2500 U/kg achieved neuroprotective serum levels.

A Comparison of High-Dose Recombinant Erythropoietin Treatment Regimens in Brain-Injured Neonatal Rats

Recombinant human erythropoietin (rEpo) is neuroprotective in neonatal models of hypoxic-ischemic brain injury. However, the optimal rEpo dose, dosing interval, and number of doses for reducing brain injury are still undetermined. We compared the neuroprotective efficacy of several subcutaneous rEpo treatment regimens. Seven-day-old rats underwent unilateral carotid ligation plus 90 min 8% hypoxia. Treatment began immediately after injury. Treatment regimens examined included 1, 3, or 7 daily subcutaneous injections of either 0 (vehicle), 2,500, 5,000, or 30,000 U/kg rEpo. Gross brain injury, neuronal apoptosis (TUNEL), and gliosis (glial fibrillary acidic protein) were assessed at 48 h or 1 wk post injury. Immunoreactive cells and brain injury were quantified for statistical comparison to vehicle controls. rEpo treatment reduced brain injury, apoptosis, and gliosis, in a dose-dependent U-shaped manner at both 48 h and 1 wk. Neither one injection of 2,500, seven injections of 5,000, or three injections of 30,000 U/kg rEpo were protective. Three doses of 5,000 and one dose of 30,000 U/kg rEpo were most protective at both time intervals. rEpo provides dose-dependent neuroprotection. Of the regimens tested, three doses of 5,000 U/kg was optimal because it provided maximal benefit with limited total exposure.

Erythropoietin Protects Dopaminergic Neurons and Improves Neurobehavioral Outcomes in Juvenile Rats after Neonatal Hypoxia-Ischemia

Brain injury as a result of hypoxia-ischemia remains a common cause of morbidity and mortality in neonates. No effective therapy is currently available. The hematopoietic cytokine erythropoietin (Epo) provides neuroprotection in many adult models of brain injury and is currently being investigated as a therapeutic agent for human stroke and spinal cord injury. We tested the hypothesis that recombinant Epo (rEpo) would improve neurobehavioral outcomes after neonatal hypoxic-ischemic brain injury. Postnatal day 7 rats underwent right common carotid artery occlusion followed by a 90-min exposure to 8% oxygen. Rats were subsequently treated with rEpo or placebo. Sensory neglect and apomorphine-induced rotation were measured at P27 and P28. Rats were killed at P30, blood was drawn, and the brains were perfusion-fixed for histology and immunohistochemistry. No differences in gross brain injury between rEpo and placebo-treated rats were found. Neonatal rEpo treatment protected dopamine neurons as indicated by the preservation of tyrosine hydroxylase–positive cells in the substantia nigra pars compacta and ventral tegmental area. rEpo treatment also improved functional outcomes by reducing sensory neglect and preventing the rotational asymmetry seen in control animals. No differences in hematocrit, white blood cell counts, neutrophil counts, or platelet counts were measured. We observed that rEpo treatment protected mesencephalic dopamine neurons and reduced the degree of behavioral asymmetries at 4 wk of life. On the basis of these findings, we conclude that further studies investigating the safety and efficacy of high-dose rEpo as a neuroprotective strategy are indicated in neonatal models of hypoxic-ischemic brain injury.

Erythropoietin Concentrations in Cerebrospinal Fluid of Nonhuman Primates and Fetal Sheep following High-Dose Recombinant Erythropoietin

Erythropoietin (Epo) decreases neuronal injury and cell death in vitro and in vivo. To lay the groundwork for use of Epo as a potential therapy for brain injury, we tested the hypothesis that systemic dosing of high-dose recombinant Epo (rEpo) would result in neuroprotective rEpo concentrations in the spinal fluid of adult and developing animals. This report characterizes the pharmacokinetics of high-dose rEpo in the blood and spinal fluid of juvenile and adult nonhuman primates (n = 7) and fetal sheep (n = 37) following a single injection. Timed blood and spinal fluid samples were collected following rEpo injection. Epo accumulation in spinal fluid was dependent on peak serum concentration and time following injection. We demonstrate that high-dose rEpo was well tolerated and results in neuroprotective concentrations in spinal fluid of adult and developing animal models by 2–2.5 h after injection.

Pharmacokinetics of High-Dose Recombinant Erythropoietin in Plasma and Brain of Neonatal Rats

Recombinant human erythropoietin (rEpo) is neuroprotective in neonatal models of brain injury. Pharmacokinetic data regarding the penetration of circulating rEpo into brain tissue is needed to optimize neuroprotective strategies. We sought to determine the pharmacokinetics of rEpo given intraperitoneally or subcutaneously in plasma and brain. We hypothesized that 1) exogenous rEpo would penetrate the blood-brain barrier (BBB), 2) brain and plasma Epo would correlate, and 3) brain injury would enhance rEpo penetration. Two hundred and eighty-four 7-d-old control, sham, or brain-injured rats were treated with i.p. or s.c. rEpo (0, 250, 2500, or 5000 U/kg) and killed at scheduled intervals. Plasma and brain tissue were collected. Epo concentrations were measured by ELISA. Intraperitoneal injection yielded a faster and greater peak concentration of plasma rEpo (Tmax 3 h, Cmax 10,016 ± 685 mU/mL) than s.c. injection (Tmax 9 h, Cmax 6224 ± 753 mU/mL). Endogenous brain Epo was below detection even after hypoxia exposure. Systemic rEpo crossed the BBB in a dose-dependent manner, peaked in brain at 10 h, and was increased after brain injury. We conclude that high-dose rEpo is detectable in brain for >20 h after a single systemic injection. These pharmacokinetic data are valuable for planning of rEpo neuroprotection experiments.

Concurrent erythropoietin and hypothermia treatment improve outcomes in a term nonhuman primate model of perinatal asphyxia.

Background: Up to 65% of untreated infants suffering from moderate to severe hypoxic-ischemic encephalopathy (HIE) are at risk of death or major disability. Therapeutic hypothermia (HT) reduces this risk to approximately 50% (number needed to treat: 7-9). Erythropoietin (Epo) is a neuroprotective treatment that is promising as an adjunctive therapy to decrease HIE-induced injury because Epo decreases apoptosis, inflammation, and oxidative injury and promotes glial cell survival and angiogenesis. We hypothesized that HT and concurrent Epo will be safe and effective, improve survival, and reduce moderate-severe cerebral palsy (CP) in a term nonhuman primate model of perinatal asphyxia. Methodology: Thirty-five Macacanemestrina were delivered after 15-18 min of umbilical cord occlusion (UCO) and randomized to saline (n = 14), HT only (n = 9), or HT+Epo (n = 12). There were 12 unasphyxiated controls. Epo (3,500 U/kg × 1 dose followed by 3 doses of 2,500 U/kg, or Epo 1,000 U/kg/day × 4 doses) was given on days 1, 2, 3, and 7. Timed blood samples were collected to measure plasma Epo concentrations. Animals underwent MRI/MRS and diffusion tensor imaging (DTI) at <72 h of age and again at 9 months. A battery of weekly developmental assessments was performed. Results: UCO resulted in death or moderate-severe CP in 43% of saline-, 44% of HT-, and 0% of HT+Epo-treated animals. Compared to non-UCO control animals, UCO animals exhibit poor weight gain, behavioral impairment, poor cerebellar growth, and abnormal brain DTI. Compared to UCO saline, UCO HT+Epo improved motor and cognitive responses, cerebellar growth, and DTI measures and produced a death/disability relative risk reduction of 0.911 (95% CI -0.429 to 0.994), an absolute risk reduction of 0.395 (95% CI 0.072-0.635), and a number needed to treat of 2 (95% CI 2-14). The effects of HT+Epo on DTI included an improved mode of anisotropy, fractional anisotropy, relative anisotropy, and volume ratio as compared to UCO saline-treated infants. No adverse drug reactions were noted in animals receiving Epo, and there were no hematology, liver, or kidney laboratory effects. Conclusions/Significance: HT+Epo treatment improved outcomes in nonhuman primates exposed to UCO. Adjunctive use of Epo combined with HT may improve the outcomes of term human infants with HIE, and clinical trials are warranted.

Erythropoietin for infants with hypoxic-ischemic encephalopathy.

Purpose of review Perinatal asphyxia, intraventricular hemorrhage and stroke are common causes of neonatal brain injury, with hypoxia–ischemia as the final common pathway of injury. Erythropoietin (Epo) has potential to lessen neurologic sequelae due to hypoxia–ischemia. The purpose of this review is to highlight new clinical trials and experimental evidence that expand our understanding of Epo as a potential treatment for perinatal brain injury. Recent findings Several trials of Epo treatment are reviewed: two phase I/II trials of high-dose Epo given to preterm infants established pharmacokinetic and safety profiles, and a trial of Epo treatment for term infants with moderate hypoxic–ischemic encephalopathy found reduced disability. Potential risks and benefits of high-dose Epo are discussed. New evidence related to Epo receptor expression, signal transduction pathways, and mechanisms of neuroprotection are reviewed. Summary Cautious optimism is warranted regarding the use of high-dose Epo as a treatment option for neonatal brain injury. To date, Epo has been well tolerated to use in neonatal populations and now studies of neuroprotective efficacy are underway.

Microarray analysis of high-dose recombinant erythropoietin treatment of unilateral brain injury in neonatal mouse hippocampus

Recombinant human erythropoietin (rEpo) is neuroprotective in neonatal models of brain injury. Proposed mechanisms of neuroprotection include activation of gene pathways that decrease oxidative injury, inflammation, and apoptosis, while increasing vasculogenesis and neurogenesis. To determine the effects of rEpo on gene expression in 10-d-old BALB-c mice with unilateral brain injury, we compared microarrays from the hippocampi of brain-injured pups treated with saline or rEpo to similarly treated sham animals. Total RNA was extracted 24 h after brain injury and analyzed using Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. We identified sex-specific differences in hippocampal gene expression after brain injury and after high-dose rEpo treatment using single-gene and gene set analysis. Although high-dose rEpo had minimal effects on hippocampal gene expression in shams, at 24-h post brain injury, high-dose rEpo treatment significantly decreased the proinflammatory and antiapoptotic response noted in saline-treated brain-injured comparison animals.

Recent trends in erythropoietin-mediated neuroprotection.

Fifteen years of evidence have established that the cytokine erythropoietin offers promise as a treatment for brain injury. In particular, neonatal brain injury may be reduced or prevented by early treatment with recombinant erythropoietin. Extreme prematurity and perinatal asphyxia are common conditions associated with poor neurodevelopmental outcomes including cerebral palsy, mental retardation, hearing or visual impairment, and attention deficit hyperactivity disorder. When high doses of erythropoietin are administered systemically, a small proportion crosses the blood–brain barrier and can protect against hypoxic–ischemic brain injury. In addition to other protective effects, erythropoietin can specifically protect dopaminergic neurons. Since reduced dopamine neurotransmission contributes to attention deficit hyperactivity disorder, this condition may be amenable to erythropoietin treatment. This review focuses on the potential application of erythropoietin as a neuroprotectant with regard to neurologic complications of extreme prematurity, including attention deficit hyperactivity disorder. Recent concerns that early erythropoietin might exacerbate the pathologic neovascularization associated with retinopathy of prematurity are addressed.

A new model of neonatal stress which produces lasting neurobehavioral effects in adult rats.

Background: During critical care in neonatal intensive care units (NICU), infants experience stressors and treatments that may produce lasting effects on adult health. An animal model simulating the NICU experience is needed to understand the impact of specific neonatal stressors. Objective: We combined approaches to develop a neonatal rat model simulating NICU stressors in order to examine the hypothesis that early stress and morphine sulfate (MS) exposure would affect development and alter adult behavior. Methods: Rat pups were exposed to stressors and given twice daily MS injections (2 mg/kg s.c.) for 5 days (postnatal days 3–7). Stress included daily maternal separation (from 08.00 to 16.00 h), hand feedings, a daily hypoxia/hyperoxia episode (100% N2 for 8 min, then 100% O2 for 4 min), and cold exposure (4°C for 20 min/day). Five treatment groups were formed: (1) ‘control control’ (dam reared and untreated); (2) control vehicle; (3) stress vehicle; (4) control morphine, and (5) stress morphine. Early growth and developmental indices were measured. Adult neurobehavioral tests were paw flick, passive avoidance, and forced swimming. Neonatal MS pharmacokinetics, neonatal and adult corticosterone levels, and adult hematocrit and blood pressure values were measured. Results: Neonatal stress significantly increased the mortality. Neonatal stress and MS treatment slowed early growth. Neonatal MS impaired passive avoidance learning and increased frequency, duration, and distance of forced swimming. There were no differences in corticosterone, hematocrit, or blood pressure values. Conclusions: This model simulates NICU stressors and enables measurement of acute physiological and long-term neurobehavioral indices. Neonatal MS treatment impaired the adult cognitive functioning.