## 4 Neuoprotective Peptides and Their Therapeutic Application in HIE
### Studies Using CARPs in Animal Models of HIE
#### 4.4.1 TAT-NEMO Binding Domain (NBD)  
NEMO binding domain (NBD) is an 11-amino acid peptide (NBD: TALDWSWLQTE?35-745; net charge \(-2\)) derived from the Nuclear Factor-\(\kappa\)(NF-\(\kappa\)) Essential Modulator (NEMO)/IKK-\(\gamma\)-Binding Domain. The NBD peptide was designed to inhibit the activation of NF\(\kappa\)B, a transcription factor involved in many stress responses. For example, NBD was originally developed to suppress inflammatory responses (e.g., IL-6, iNOS, MMP-9 and COX-2) and the regulation of apoptotic Bcl proteins (e.g., Bcl-2 and Bcl-x) following cytokine stimulation in several disorders including inflammatory bowel disease, arthritis, type I diabetes, multiple sclerosis and Parkinson's disease [102]. Activation of NF-\(\kappa\)B requires upstream kinase complex formation (I\(\kappa\)B-kinase; IKK) composed of the IKK\(\alpha\), IKK\(\beta\) and NEMO proteins. For NF-\(\kappa\)B activation, NEMO interacts with a carboxyl terminal sequence within both IKK-\(\alpha\) and IKK-\(\beta\), known as the NBD. The NBD peptide inhibits NF-\(\kappa\)B activation by blocking NEMO/IKK complex formation [103]. The first study examining the neuroprotective efficacy of the NBD peptide in a model of HIE (P12 rat) administered the peptide [(14,820 nmol/kg: intraperitoneally (IP)] immediately, 6 and 12 h after hypoxia and did not demonstrate any neuroprotection; instead NBD appeared to increase brain injury [104]. To improve delivery to the brain, the NBD peptide was fused to TAT (TAT-NBD: YGRKKRRQRRR-TALDWSWLQTE; net charge +6) and assessed in a P7 rat model of HIE [105], where administration (6917 nmol/kg: IP) immediately and 3 h, or as a single injection at 0, 3 or 6 h, after hypoxia was neuroprotective up to 6 weeks post-HI. When TAT-NBD was administered immediately and 3 h after hypoxia it prevented up-regulation of p53 and activation of caspase-3 24 h post-HI. TAT-NBD treatment also prevented the activation of IKK in the brain 3 h post-HI. Despite TAT-NBD inhibiting IKK and NF-\(\kappa\)B activation, it had no effect on HI-induced increases in cytokines (e.g., IL-1\(\beta\), TNF-\(\alpha\), IL-4, IL-10 and IL-1-RA). The study also assessed a TAT-NBD control peptide, where two tryptophan residues were substituted for two alanine residues (YGRKKKKRRQRRR-TALDASALQTE; net charge +6) and was shown not to be neuroprotective. Further assessment of TAT-NBD (6817 nmol/kg: IP) involving administration immediately and 3 h, immediately, 3, 6 and 12 h, immediately, 6 and 12 h, as well as 18 and 21 h after hypoxia [106], revealed long-term neuroprotection only in animals treated immediately and 3 h post-HI. Concomitantly, TAT-NBD treatment immediately and 3 h after hypoxia significantly reduced HI-mediated cytokine levels, whereas immediate, 3, 6 and 12-h treatment did not reduce cytokines. In a subsequent study, TAT-NBD treatment (6818 nmol/kg: IP) immediately and 3 h after hypoxia demonstrated significant improvements in behavioral outcomes up to 14 weeks post-HI [107]. Since intrauterine infections can be associated with HIE, TAT-NBD was assessed in a lipopolysaccharide-sensitized (IP injection) HIE model in the P7 rat [108]. Animals were administered with TAT-NBD (477 nmol/kg) intranasally (IN) 10 min after hypoxia, using three HI injury conditions; HI only, 4-h LPS-pre-treatment + HI or 72-h LPS-pre-treatment + HI. Treatment with TAT-NBD significantly reduced brain injury only in the LPS-sensitized HI models, when assessed 7 days post-HI. While it was surprising that TAT-NBD did not reduce brain injury following HI only, its neuroprotective actions in LPS-sensitized HI models may be due to the ability of the cationic TAT-NBD peptide to electrostatically bind and neutralize anionic LPS molecules or by reducing the effects of inflammatory cytokines. Neutralizing LPS would have the effect of suppressing the molecules capacity to evoke an immune response and inflammatory response.