Efficacy and tolerability of the galanin analog NAX 5055 in the multiple-hit rat model of symptomatic infantile spasms

Abstract

Infantile spasms are seizures manifesting in infantile epileptic encephalopathies that are associated with poor epilepsy and cognitive outcomes. The current therapies are not always effective or are associated with serious side effects. Early cessation of spasms has been proposed to improve long-term outcomes. To identify new therapies for infantile spasms with rapid suppression of spasms, we are using the multiple-hit rat model of infantile spasms, which is a model of refractory infantile spasms. Here, we are testing the efficacy and tolerability of a single dose of the galanin receptor 1 preferring analog, NAX 5055, in the multiple-hit model of spasms. To induce the model, postnatal day 3 (PN3) male Sprague-Dawley rats underwent right intracerebral infusions of doxorubicin and lipopolysaccharide; p-chlorophenylalanine was then injected intraperitoneally (i.p.) at PN5. After the onset of spasms at PN4, 11-14 rats/group were injected i.p. with either NAX 5055 (0.5, 1, 2, or 4mg/kg) or vehicle. Video monitoring for spasms included a 1h pre-injection period, followed by 5h of recording post-injection, and two 2h sessions on PN5. The study was conducted in a randomized, blinded manner. Neurodevelopmental reflexes were assessed daily as well as at 2h after injection. Respiratory function, heart rate, pulse distension, oximetry and blood glucose were measured 4h after injection. The relative expression of GalR1 and GalR2 mRNA over β-actin in the cerebral cortex and hippocampus was determined with real time reverse transcription polymerase chain reaction. There was no acute effect of NAX 5055 on spasm frequency after the single dose of NAX 5055 (n=11-13 rats/group, following exclusions). Neurodevelopmental reflexes, vital signs, blood glucose measured 4h post-injection, and survival were not affected. A reduction in pulse and breath distention of unclear clinical significance was observed with the 7mg/kg NAX 5055 dose. GalR1 mRNA was present in the cerebral cortex and hippocampus of PN4 and adult rats. The hippocampal - but not the cortical - GalR1 mRNA expression was significantly lower in PN4 pups than in adults. GalR1 mRNA was also at least 20 times less abundant in the PN4 cortex than GalR2 mRNA. In conclusion, a single dose of NAX 5055 has no acute efficacy on spasms or toxicity in the multiple hit rat model of medically refractory infantile spasms. Our findings cannot exclude the possibility that repetitive NAX 5055 administration may show efficacy on spasms. The higher expression of GalR2 in the PN4 cortex suggests that GalR2-preferring analogs may be of interest to test for efficacy on spasms.

Keywords: ACTH; Antibody; Antiepileptic; Cerebral cortex; GalR; Galanin receptor; Glucose; IS; NG; Neurodevelopmental reflexes; OFA; PN; SRT; adrenocorticotropic hormone; galanin receptor; i.p.; infantile spasms; intraperitoneal; negative geotaxis; open field activity; postnatal day; surface righting time.

Figure 1. A single dose of NAX 5055 given after the onset of spasms does not suppress spasms in the multiple-hit model (PN4 and PN5)

Panel A. Hourly raw frequencies of spasms after a single injection of NAX 5055 or its vehicle at PN4 in pups treated according to the multiple-hit protocol. NAX-0.5 group had more frequent spasms compared with the VEH group at the 4^th post-injection hour. NAX-4 group had less spasms compared to the other NAX groups but not compared to the VEH, at the 4^th hour post-injection. Panel B. Normalized frequencies of spasms (% of pre-injection frequencies of the same rat) after a single injection of NAX 5055 or its vehicle in pups treated according to the multiple-hit protocol. No significant differences were found among treatment groups. Please see also Tables 1 and 2 for the statistics. Bars represent standard deviation. NAX 5055 dose groups are as follows: NAX-0.5 = 0.5 mg/kg, NAX-1 = 1mg/kg, NAX-2 = 2mg/kg, NAX-4 = 4 mg/kg, VEH = vehicle. Panel C. The table depicts P values derived from ANOVA comparisons of raw and normalized frequencies of spasms among the different treatment groups at each time point. Only the P-value of the raw frequencies during the 4^th post-injection hour is statistically significant. Pairwise comparisons of the mean raw frequencies of spasms among the different doses showed less frequent spasms in the NAX-4 group compared to each of the other NAX 5055 doses, but not compared to the VEH.

Figure 2. NAX 5055 has no significant effect on neurodevelopmental reflexes

Panels A–C. SRT (panel A), OFA (panel B), and NG (panel C) were tested daily in the morning (PN3, PN4AM, PN5) as well as at 2 hours following the NAX 5055 injection (PN4PM). Bars represent standard deviation. Panel D. The table presents the P values derived from ANOVA comparisons of mean scores for SRT, OFA, and NG among the different treatment groups at each timepoint. No significant differences among groups were seen in these scores.

Figure 3. Expression of GalR1 and GalR2 mRNA in PN4 rat cerebral cortex

Panel A: Quantitative RT-PCR of total RNA extracts from cerebral cortex of male PN4 pups (controls or pups treated according to the multiple-hit protocol) revealed significantly more GalR2 mRNA compared to GalR1 mRNA (Gene effect: F_{(7, 23)}= 40.45, P<0.0001; n=3 rats per group). There were no inter-hemispheric or treatment-related differences in GalR1 or GalR2 mRNA expression. Results are expressed as 10⁻⁶ * (β-actin mRNA). Panel B: The DNA products of the qRT-PCR reactions for GalR1 (115 bp) and GalR2 (116 bp) from right cerebral cortical RNA samples were separated on a Tris-borate-EDTA/ ethidium bromide gel. RT-PCR was performed using 300ng of total RNA for the GalR1 assay and 100ng total RNA for the GalR2 assay. Panel C: GalR1 mRNA was detected using qRT-PCR in the anterior dorsal hippocampus of both PN4 control rats and rats treated according to the multiple-hit model, but at low levels (n= 3 rats per group). The right hippocampi of the multiple-hit PN4 rats have higher GalR1 mRNA expression compared with the right hippocampi of the controls (Kruskall-Wallis, P<0.05). Black bars indicate right and grey bars the left hippocampal samples. Panel D: Expression of GalR1 mRNA expression in the cortex and anterior dorsal hippocampus of PN4 and adult control rats, using qRT-PCR. No significant developmental differences are found in the sensorimotor cortex. In contrast, GalR1 mRNA expression increased in the hippocampus of adult control rats compared with PN4 rats (Kruskall-Wallis test, P<0.05; n=3 rats/group; 2 hippocampi per rat). Panel E: Gel electrophoresis of the qRT-PCR products for GalR1 (115bp) and β-actin (91 bp) from the right hippocampus of nine PN4 and adult rats. Bars indicate the standard deviations of the mean. The asterisks indicate statistical significance P<0.05. CCX: cerebral cortex; R: right; L: left; Hippo: hippocampus.