Short-Term Dose–Response Characteristics of 2-Iminobiotin Immediately Postinsult in the Neonatal Piglet After Hypoxia-Ischemia
Background and Purpose—To determine the optimal dose of 2-iminobiotin (2-IB) for the treatment of moderate to severe asphyxia in a neonatal piglet model of hypoxia-ischemia.
Methods—Newborn piglets were subjected to a 30-minute hypoxia-ischemia insult and randomly treated with vehicle or 2-IB (0.1 mg/kg, 0.2 mg/kg, or 1.0 mg/kg). aEEG background and seizure activity were scored after hypoxia-ischemia every 4 h until 24 h and at 48 h and neurobehavioral scores were obtained. Brain tissue was collected and processed for analysis of caspase-3 activity, histology, and tyrosine nitration.
Results—A dose range of 0.1 to 1.0 mg/kg/dose of 2-IB improved short-term outcome as demonstrated by an increased survival with a normal aEEG and decreased nitrotyrosine staining in the 2-IB–treated animals, indicating decreased cellular damage. Neurobehavior, caspase-3 activity in thalamus, and histology scores were not significantly different.
Conclusions—Based on survival with a normal aEEG, 0.2 mg/kg 2-IB is likely to be the most appropriate dose for use in future clinical trials in neonates with perinatal hypoxia-ischemia.
Perinatal hypoxia-ischemia (HI) is a significant cause of neonatal brain injury. Neonatal animal models of HI show that excessive production of nitric oxide (NO), mediated by nitric oxide synthases (NOS), play an important role in the pathogenesis of neuronal injury after HI in the neonate.1,2 Three isoforms of NOS exist: the constitutively expressed neuronal NOS, endothelial NOS, and the inducible NOS. In vitro studies have shown that selective inhibition of neuronal NOS and inducible NOS can be achieved by the NOS inhibitor 2-iminobiotin (2-IB).3
To transition treatment to the human term neonate, it is important to know the dose–response effect of 2-IB to identify the optimal dose to be given after perinatal HI. The aim of this study was to determine the dose–response characteristics of 2-IB in a preclinical animal model of perinatal HI and to establish the most effective dose (range) for future clinical trials. This study was performed in our piglet model of inhalational HI, which is clinically, electrophysiologically, and neuropathologically comparable with the term born human neonate.4
Please see the online-only Data Supplement for expanded Methods. Experiments were performed in accordance with National Health and Medical Research Council guidelines (Australia) and approved by the University of Queensland Animal Ethics Committee.
The HI insult was performed in term neonatal piglets (n=47) as previously described5; 6 animals served as sham-operated controls. HI piglets were randomly assigned to blinded treatment with vehicle or 2-IB at 0.1, 0.2, or 1 mg/kg/dose i.v. immediately post-HI and dosing repeated every 4 h until 20 h (6 doses in total). aEEG background pattern, presence of epileptic activity, and neurobehavior were scored.6
At 48 h postinsult animals were euthanized and tissue analyzed for caspase-3 activity, tyrosine nitration, and histology (see the online-only Data Supplement).
In total, 47 piglets were subjected to HI; 16 piglets were only mildly affected (continuous normal voltage at 30 min post-HI; see the online-only Data Supplement for examples) and excluded from further analysis. Of the remaining 31 piglets, 10 were vehicle-treated, 7 received 0.1 mg/kg/dose, 9 received 0.2 mg/kg/dose, and 5 received 1.0 mg/kg/dose. There was no difference in birth weight, postnatal age, pH, arterial BE, PO2, PCO2, duration of hypotension, heart rate, or temperature between treatment groups (see the online-only Data Supplement).
There was a significant overall effect of 2-IB on survival with a normal aEEG at 48 h (P=0.0047; Table 1). Treatment was effective in all 3 dosing groups, with the 0.2 mg/kg/dose group showing the highest proportion of surviving animals with a normal aEEG at 48 h. 2-IB treated piglets also demonstrated significantly less tyrosine nitration in thalamus, parietal, and temporal cortex at all doses versus vehicle-treated piglets (Figure). No nitrotyrosine-modified substrates were observed in sham-operated piglets.
Caspase-3 activity in thalamus was not significantly different between groups (P=0.096; Table 2). aEEG-patterns over time are shown in the online-only Data Supplement. Electrographic seizure activity was detected in all HI-injured piglets from about 4 h onwards until 48 h but was abolished in the 0.2 and 1.0 mg/kg dose groups between 24 and 48 h. Neurobehavioral and histology scores did not show significant differences (see the online-only Data Supplement).
The aim of the present study was to determine the short-term dose–response characteristics of 2-IB for treatment of moderate to severe perinatal HI. Animals treated with 2-IB demonstrated greater survival with a normal aEEG at 48 h and reduced tyrosine nitration. Decreased nitrotyrosine staining supports the NO pathway as a potential mechanism of neonatal HI injury. The aEEG background pattern is known to be an early predictor of brain injury in term infants with hypoxic-ischemic encephalopathy.7 Because piglet HI brain injury closely mimics that of human HI brain injury, the effect of 2-IB on aEEG background pattern can be considered a good biomarker of clinical outcome.8 In our piglet model, 2-IB treatment promoted recovery of aEEG background pattern and reduced epileptic activity.
In human term-equivalent rats, 2-IB neuroprotection has been demonstrated at 10 mg/kg/dose when administered s.c. three times in 24 h.9 Previous studies in newborn piglets examined only 0.2 mg/kg/dose and reported neuroprotection at 24 h with 6 doses (i.v.) every 4h.10,11 Our current study supports that the full dose range of 0.1 to 1.0 mg/kg 2-IB is safe; the 0.2 mg/kg/dose shows the most promising short-term outcome data in our piglet model of perinatal HI. Limitations of this study include the treatment with 2-IB immediately postinsult, and the potential bias caused by the exclusion of mildly affected piglets. Because hypothermia is currently an established treatment for perinatal HI in high-income countries, preclinical studies of combination therapy of 2-IB with delayed hypothermia would be essential before a clinical trial can be considered. For translation into human clinical studies (perinatal HI without hypothermia), we believe that a starting dose comparable with the 0.2 mg/kg dose in the piglet will be optimal and safe.
We thank Stephanie Miller and Dr Ir. Mira Wenker.
Sources of Funding
Funding support was provided from Neurophyxia B.V., National Health and Medical Research Council (Australia), and UQ Center for Clinical Research.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.112.677922/-/DC1.
- Received September 24, 2012.
- Accepted December 6, 2012.
- © 2013 American Heart Association, Inc.
- Thoresen M,
- Hellström-Westas L,
- Liu X,
- de Vries LS
- van den Tweel ER,
- van Bel F,
- Kavelaars A,
- Peeters-Scholte CM,
- Haumann J,
- Nijboer CH,
- et al
- Peeters-Scholte C,
- Koster J,
- Veldhuis W,
- van den Tweel E,
- Zhu C,
- Kops N,
- et al