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.

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. (Pediatr Res 61: [451][452][453][454][455] 2007) N eonatal brain injury continues to affect the lives of thousands of infants in the United States and worldwide. In fact, perinatal asphyxia accounts for 23% of deaths globally (1). Hypothermia following hypoxia-ischemia offers promise of improved neurodevelopmental outcomes, however, thus far, the therapeutic benefit has been modest (2). When strict entry criteria are defined, up to 60% of untreated infants exposed to moderate to severe perinatal hypoxia-ischemia die or have significant neurodevelopmental sequelae (3,4). Erythropoietin (Epo), a hematopoietic cytokine known best for its erythropoietic effects, has neuroprotective effects in many animal models of brain injury, including hypoxia-ischemia, trauma, and excitotoxic injury (5-12). Although many neuroprotective strategies appeared promising in animal models, most have failed clinically (often due to toxicity). In contrast, rEpo is a safe treatment for anemia (13) and, more importantly, the neuroprotective effects of rEpo have translated well from bench to clinical trials for both amyotrophic lateral sclerosis (ALS) (14) and middle cerebral artery stroke (15).Initial studies provided no evidence that the 37 kD charged glycoprotein rEpo could cross the blood-brain barrier (BBB) (16 -18). Therefore, early rEpo neuroprotection experiments used either in vitro application (19,20) or direct intracerebral injection (21). More recently, robust neuroprotection was produced after systemic injection of high-dose rEpo (5,000 U/kg i.p.) (5). Although the neuroprotective rEpo dose is clearly above the anemia treatment range, there is no consensus as to the optimal neuroprotective dose, dose number, or in...

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.

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 Ϯ 685 mU/mL) than s.c. injection (Tmax 9 h, Cmax 6224 Ϯ 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 highdose rEpo is detectable in brain for Ͼ20 h after a single systemic injection. These pharmacokinetic data are valuable for planning of rEpo neuroprotection experiments.

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.

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 placebotreated 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. Abbreviations ADHD, attention-deficit/hyperactivity disorder DA, dopamine Epo, erythropoietin EpoR, erythropoietin receptor HI, hypoxia-ischemia P, postnatal day rEpo, recombinant Epo SNpc, substantia nigra pars compacta TH, tyrosine hydroxylase VTA, ventral tegmental area Perinatal exposure to hypoxia-ischemia (HI) produces brain injury that is a significant source of morbidity and mortality for preterm and term neonates. Neonatal HI exposure initiates a multifactorial cascade that can persist for days, producing injury to multiple brain regions with corresponding motor, behavioral, and cognitive impairments. Although there are currently no effective therapies to ameliorate hypoxic-ischemic brain injury, several useful rodent models of neonatal brain injury are available. Using 7-d-old (P7) rat pups, which are comparable to near-term humans in brain maturity (1), the technique entails unilateral ligation of the common carotid artery and subsequent exposure of the animals to hypoxia (Rice-Vannucci model) (2). This procedure produces permanent unilateral brain injury in multiple regions, with specific impact on the basal ganglia including decreases in striatal dopamine (DA) receptors and increases in striatal DA transporters (3,4). Disruption of dopaminergic neurotransmission is suspect for several de...

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 braininjured 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-

Objective To evaluate long-term outcomes of 60 extremely low birth weight (ELBW) infants treated with or without three injections of high-dose erythropoietin (Epo). Study Design A retrospective analysis of anthropometric and neurodevelopmental outcome data comparing 30 ELBW infants enrolled in a phase I/II study examining the pharmacokinetics of high-dose Epo (500, 1000 and 2500 U/kg × 3 doses) administered to 30 concurrent controls. Result Birth characteristics and growth from 4 to 36 months were similar for untreated and Epo-treated patients. Multiple linear regression analysis of neurodevelopmental follow-up scores from 17/25 Epo-treated and 18/26 control infants identified that Epo correlated with improvement of cognitive (R = 0.22, P = 0.044) and motor (R = 0.15, P = 0.026) scores. No negative long-term effects of Epo treatment were evident. Conclusion Retrospective analysis of the only available long-term follow-up data from ELBW infants given high-dose Epo treatment suggests that Epo treatment is safe and correlates with modest improvement of neurodevelopmental outcomes.