Melatonin and/or erythropoietin combined with hypothermia in a piglet model of perinatal asphyxia

Abstract

As therapeutic hypothermia is only partially protective for neonatal encephalopathy, safe and effective adjunct therapies are urgently needed. Melatonin and erythropoietin show promise as safe and effective neuroprotective therapies. We hypothesized that melatonin and erythropoietin individually augment 12-hour hypothermia (double therapies) and hypothermia + melatonin + erythropoietin (triple therapy) leads to optimal brain protection. Following carotid artery occlusion and hypoxia, 49 male piglets (<48 hours old) were randomized to: (i) hypothermia + vehicle (n = 12), (ii) hypothermia + melatonin (20 mg/kg over 2 hours) (n = 12), (iii) hypothermia + erythropoietin (3000 U/kg bolus) (n = 13) or (iv) triple therapy (n = 12). Melatonin, erythropoietin or vehicle were given at 1, 24 and 48 hours after hypoxia-ischemia. Hypoxia-ischemia severity was similar across groups. Therapeutic levels were achieved 3 hours after hypoxia-ischemia for melatonin (15-30mg/L) and within 30 minutes of erythropoietin administration (maximum concentration 10,000 mU/mL). Compared to hypothermia + vehicle, we observed faster amplitude integrated EEG recovery from 25-30 hours with hypothermia + melatonin (p = 0.02) and hypothermia + erythropoietin (p = 0.033) and from 55-60 hours with triple therapy (p = 0.042). Magnetic Resonance Spectroscopy Lactate/N-acetyl aspartate peak ratio was lower at 66 hours in hypothermia + melatonin (p = 0.012) and triple therapy (p = 0.032). With hypothermia + melatonin, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells were reduced in sensorimotor cortex (p = 0.017) and oligodendrocyte transcription factor 2 labelled-positive counts increased in hippocampus (p = 0.014) and periventricular white matter (p = 0.039). There was no reduction in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells with hypothermia + erythropoietin, but increased oligodendrocyte transcription factor 2 labelled-positive cells in 5 of 8 brain regions (p < 0.05). Overall, melatonin and erythropoietin were safe and effective adjunct therapies to hypothermia. Hypothermia + melatonin double therapy led to faster amplitude integrated EEG recovery, amelioration of Lactate/N-acetyl aspartate rise and reduction in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells in the sensorimotor cortex. Hypothermia + erythropoietin double therapy was association with EEG recovery and was most effective in promoting oligodendrocyte survival. Triple therapy provided no added benefit over the double therapies in this 72-hour study. Melatonin and erythropoietin influenced cell death and oligodendrocyte survival differently, reflecting distinct neuroprotective mechanisms which may become more visible with longer term studies. Staggering the administration of therapies with early melatonin and later erythropoietin (after hypothermia) may provide better protection; each therapy has complementary actions which may be time critical during the neurotoxic cascade after hypoxia-ischemia.

Melatonin for Neonatal Encephalopathy: From Bench to Bedside

“…Initial pilot studies using Sigma-Aldrich melatonin, diluted in ethanol (2.5% v / v ) given intravenously at infusion rates ≥10 mg/kg/h was associated with hypotension requiring 0.9% saline boluses and inotropic support [ 98 ]. However, this was not observed in our most recent study using the Chiesi ethanol-free melatonin formulation given intravenously at 10 mg/kg/h [ 101 ]. It is unclear whether ethanol contributed to the observed fall in blood pressure in the pilot studies; animal studies are conflicting.…”

“…More rapid melatonin administration (18 mg/kg over 2 h) in the Robertson et al, 2020 [ 100 ] study, achieving target therapeutic levels (18.84 mg/L) earlier (within 3 h of HI), was associated with aEEG recovery from 19 h, reduction in Lac/NAA peak ratios in the white matter and thalamus and overall reduction in TUNEL positive cells compared to HT alone. Although the ethanol excipient likely added to the neuroprotective action of the melatonin solution in this study [ 100 ], our subsequent study (Pang et al 2021) [ 101 ] using Chiesi melatonin at a higher dose of 20 mg/kg given over 2 h at 1 h after HI, repeated at 24 h and 48 h, augmented 12 h HT in all three primary outcome measures. In HT + melatonin animals, cerebral background aEEG recovery was more rapid from 25–30 h, Lac/NAA peak ratio reduced in the thalamus at 66 h, and regional reduction in TUNEL positive cells in the sensorimotor cortex was observed compared to HT + vehicle.…”

“…The safety profile of melatonin doses of up to 30 mg/kg in ethanol [ 98 , 115 ] and ethanol-free formulations [ 99 , 101 ] (Chiesi Int. pat.…”

“…In HT + melatonin animals, cerebral background aEEG recovery was more rapid from 25–30 h, Lac/NAA peak ratio reduced in the thalamus at 66 h, and regional reduction in TUNEL positive cells in the sensorimotor cortex was observed compared to HT + vehicle. In the absence of the confounding effects of ethanol, both studies using Chiesi melatonin [ 99 , 101 ] reported consistent localised protection in the sensorimotor cortex, a region of high metabolic activity [ 121 ] and therefore, susceptible to high levels of oxidative stress during energy failure in HI. The C max achieved was higher (27.9 mg/L) and earlier (3 h) compared to the original Robertson et al, 2013 [ 98 ] study, but trough levels prior to the second dose were lower (10 mg/L vs. 17 mg/L) ( Figure 3 ).…”

The Neonatal and Juvenile Pig in Pediatric Drug Discovery and Development

“…An excess of nitric oxide is produced in the first hours after birth, which causes brain cell damage. In this regard, EMA approved a PIP using newborn pigs as a well-established model of PA [ 26 , 155 , 156 , 157 ] with the nitric oxide synthase inhibitor 2-iminobiotin. This drug was approved as an orphan drug as it reduces brain damage in the first 24 h after birth [ 158 ].…”