NNZ-2566 in Rett Syndrome and Autism Spectrum Disorders Role and Update 1
Overview The natural growth factor IGF-1 is broken down in the body to IGF-1[1-3] NNZ-2566 is an analogue of IGF-1[1-3] developed by Neuren Pharmaceuticals Ltd. NNZ-2566 has enhanced oral availability and a pharmaceutical profile suitable for investigation in autism spectrum disorders. Autism, Rett Syndrome and Fragile X Syndrome: disorders of synaptic connectivity involving neuroinflammation IGF-1[1-3] and NNZ-2566 have benefit in models of Rett Syndrome and Fragile X Syndrome Clinical study in Rett Syndrome is underway Clinical study in Fragile X Syndrome is being planned 2
IGF-1, IGF-1[1-3] and NNZ-2566 3
IGF-1 and IGF-1[1-3] IGF-1 is essential for brain development, widely expressed in the CNS, upregulated following brain injury Naturally occurring neurotrophic factor one of the brain s self-repair mechanisms IGF-1[1-3] is believed to be the active neuroprotective moiety in response to injury IGF-1 [1-3]appears to upregulate IGF-1 in the brain, enhance IGF1R phosphorylation and expression of synaptic markers 4
IGF-1[1-3] Mechanism of Action IGF-1[1-3]: Reduces cytokines 2 and neuroinflammatory markers in brain Activates Akt-mToR pathway in microglia Increases markers of presynaptic and postsynaptic synapses Activates Akt-mToR pathway in mecp2 knockout mouse model of Rett Syndrome IGF[1-3] reduces number of microglia in hippocampus following hypoxia ischemia in rat brain Casandra et al (2011) http://www.conferenceservices.net/reports/template/onetextabstract.xml?xsl=template/onetextabstract.xsl&abstractid=529747 Guan et al (2004) Neuropharmacology 47:892 Corvin et (2012) Neurosci Lett. 520:51 Tropea et al. (2009) PNAS 106:2029 5
NNZ-2566 (oral formulation) NNZ-2566 is a (1-3)IGF-1 analog Longer half-life Crosses blood brain barrier Orally available (45-50%) Conventional solution phase synthesis Validated manufacturing process Excellent stability Targets: inflammation, synaptic function, and microglial activation Dose-dependent effects in animal models with delayed administration No clinical toxicity in rats at highest dose tested for 28 days No SAEs, no clinically significant AEs in Phase I trial up to 100 mg/kg b.i.d. x 5 days Formulated with water Peak plasma level at 2 hours with or without food Creates opportunities for chronic dosing 6
Autism, Rett Syndrome and Fragile X Syndrome 7
Synapsopathy in ASDs Altered synapses in idiopathic (non-syndromal) and syndromal autism Functional consequences: impairments in synaptic plasticity, excitatory/inhibitory imbalance, and disrupted neuronal synchrony Hutsler and Zhang (2010) Brain Res 1309:83 Irwin et al (2000) Cerebral Cortex 10:1038 Chapleau et al (2009) Neurobiol Dis 35:219 8
Synapsopathy in ASDs Dolan and Bear, J Neurodev Disord 2009 June; 1(2): 133 140
ASDs: impaired neuronal network performance Kelleher and Bear, Cell 135, October 31, 2008
Neuroinflammation in ASDs Microglia and astroglia are activated in brain in autism Fragile X Syndrome astrocytes can institute neuronal phenotype, and wild type astrocytes can rescue Fragile X Syndrome phenotype Wild type microglia rescue Rett Syndrome phenotype Vargas et al (2005) Ann Neurol. 57:67 Jacobs et al (2010) BMC Neurosci. 11:132 Maezawa and Jin (2010) J Neurosci. 30:5346 11
Cytokines in ASDs Cytokines are cell signalling molecules produced by immune system cells including microglia Interleukin-6 is an example: Interleukin-6 may be involved in autism, Fragile X Syndrome and Rett Syndrome Interleukin-6 can activate microglia IL-6 induces changes in dendritic spine density, alters neuronal adhesion and migration, causes excess in excitatory synapses, and reduces social interaction in an animal model of autism Ashwood et al (2011) Brain Behav Immun. 25:40 Ashwood et al (2010) Brain Behav Immun. 24:898 De Filippis et al (2012) Neuropsychopharmacology 37:1152 Krady et al (2008) J Neurosci Res. 86:1538 Wei et al (2012) Biochim Biophys Acta. 1822:831 Wei et al (2011) J Neuroinflammation 8:52 12
Glia-neuron interactions 13 Blank and Prinz, Glia 61:62-70 (2013)
Neuronal Transcriptional Control, Growth and Survival: Cytokines and Growth Factors in Fragile X Syndrome Effect of NNZ-2566 NNZ-2566 Believed to attenuate aberrant cytokine levels IL-6 GP130 Activated Microglia NNZ-2566 Believed to restore normal microglia function; perhaps through normalizing autocrine homeostasis Caspase-8 Bid Bax Caspase-3 STAT3 JAK3 mtor PI3K Akt NF- κb Protein Synthesis Regulation Normalised Dendritic Growth NNZ-2566 Reverses Akt in fmr1 KO _ mice Bad Ras MEK ERK NNZ-2566 Reverses ERK in fmr1 KO mice Normal Dendritic Pruning Cell Death / Apoptosis
Neuronal Transcriptional Control, Growth and Survival : Cytokines and Growth Factors in Rett Syndrome Effect of NNZ-2566 NNZ-2566 Believed to attenuate aberrant cytokine levels IGF-1(1-3) reported to increase IGF-1 receptor activation; may help reverse impaired Akt/mTOR activation in absence of MeCP2 IL-6 GP130 Restoration of Microglia NNZ-2566 Hypothesized to restore normal function of microglia. Increased microglial Atf3 is reported, this would reduce local IL-6 and TNF-α, attenuating autocrine activation. IGF-1(1-3) reported to activate Akt in microglia Caspase-8 Bid Bax Caspase-3 STAT3 JAK3 mtor PI3K Akt NF- κb _ Bad Ras MEK ERK Increase in total brain pakt reported mice following IGF-1(1-3) Protein Synthesis Regulation Improved Dendritic Growth Improved Dendritic Pruning Cell Death / Apoptosis
Mechanism of Action Summary NNZ-2566 is not a direct agonist of the IGF-1 receptor Traumatic brain injury Microglial activation and cytokine release stimulates pro-apoptotic pathways NNZ-2566 reduces neuroinflammation and apoptosis Fragile X Syndrome Activation of astroctyes induces Fragile X Syndrome neuronal phenotype, potentially via induction of RAS-MEK-ERK and PI3K-Akt-mToR pathways NNZ-2566 reverses neuroinflammation and Akt / ERK activation Rett Syndrome Impaired passive sentinel function of microglia related to PI3k-Akt-mToR function NNZ-2566 reverses Akt dysfunction normalizing microglial support of synaptic function 16
Effects on cytokines in brain injury 17
Normalized mrna level Time - Course of Gene Expression (qrt-pcr) 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0.6 0.5 0.4 0.3 0.2 0.1 1 1 1 0 0 1 1 IL-1b 1 1 1 1 1 1 1 Sham 1hr 4hr 12hr 24hr 72hr 7ds IL-6 # # 1 1 2 1 Sham PBBI PBBI+NNZ-2566 Sham 1hr 4hr 12hr 24hr 72hr 7ds J Neuroinflammation (2009) # # Time Post-PBBI 0.5 0.4 0.3 0.2 0.1 0 0.3 0.25 0.2 0.15 0.1 0.05 0 TNF-a 1 1 E-selectin # # # Sham 1hr 4hr 12hr 24hr 72hr 7ds # Sham 1hr 4hr 12hr 24hr 72hr 7ds 18 #
Effects in Fragile X Syndrome model 19
Rescue of dendritic spines fmr1 KO mouse neurons show increased numbers of dendritic spines NNZ-2566 in the concentration range 0.5 to 50 nm dose dependently reduces excess spine number 20
Open Field The open field is an exposed space in which movement can be tracked. During exposure to the open field mice will habituate to the environment and thus explore less, decreasing the amount movement they show over time. The present experiment records movement and rearing during an initial exposure, during a second exposure after 10 minutes and during a third exposure after 24hours. Failures to reduce locomotion or rearing at 10 minutes and 24 hours indicate deficits in short and long term memory, respectively. A variety of measures can be collected in the open field locomotor activity and rearing are commonly reported
Rears / 3 min Open Field Rearing 35 Wild Type Vehicle 30 Wild Type NNZ-2566 25 p < 0.01 p < 0.01 fmr1 KO Vehicle fmr1 KO NNZ-2566 20 15 NS p < 0.01 N = 10 per group 10 NS 5 NS 0 0 Trial 1 Trial 2 Trial 3 fmr1 KO mice show increased activity NNZ-2566 has no effect in Wild Type mice, but significantly reduces rears in fmr1 KO mice fmr1 KO mice do not show habituation at 10 min (short term memory deficit) Thus during the short term and long term memory tests, NNZ-2566 treated fmr1 KO mice do not differ from controls
Elevated Plus Maze Time Spent The elevated plus maze has two open and two closed arm, raised above floor height. The open arms are more exposed and therefore create more anxiety in the mice. Mice therefore spend more time in the closed arms and visit them more. Measures taken include time spent in the arms and center of the maze, and number of arm entries
TimeSepnt Open Arm (sec) TimeSepnt Center (sec) TimeSepnt Closed Arm (sec) Elevated Plus Maze Time Spent NS NS NS p < 0.0001 0.01 p p < < 0.0001 0.01 p < p 0.0001 < 0.05 NS p < 0.0001 0.01 60 50 40 30 20 10 0 Vehicle NNZ-2566 Vehicle NNZ-2566 Wild Type frm1 KO N = 10 per group 80 70 60 50 40 30 20 10 0 NS p < 0.01 0.0001 Vehicle NNZ-2566 Vehicle NNZ-2566 Wild Type frm1 KO 300 250 200 150 100 50 0 p < 0.05 p < 0.0001 p < 0.01 Vehicle NNZ-2566 Vehicle NNZ-2566 Wild Type frm1 KO For time spent in both Open and Closed arm, fmr1 KO mice show a significant difference to Wild Type controls, as well as time spent in the center This reflects hyperactivity in the fmr1 KO mice, which spend much more time transiting the maze, compared to Wild Type mice, who remain in the closed arms This effect is significantly reversed following treatment with NNZ-2566
Social Behavior Bouts of Sniffing p < 0.001 p < 0.001 16 14 12 10 8 6 4 2 0 p < 0.01 NS Trial 1 Trial 1 Trial 1 Trial 1 Vehicle NNZ-2566 Vehicle NNZ-2566 Wild Type frm1 KO N = 10 per group fmr1 KO mice engage in significantly more bouts of sniffing compared to Wild Type mice NNZ-2566 has no significant effect in Wild Type mice After NNZ-2566 administration to fmr1 KO mice, this group is not significantly different to NNZ-2566 Wild Type mice i.e. NNZ-2566 normalizes the abnormalities in social behavior seen in fmr1 KO mice
Tests Weight (mg) Macro-orchidism p < 0.001 p < 0.001 25.0 20.0 NS NS 15.0 10.0 5.0 0.0 Vehicle NNZ-2566 Vehicle NNZ-2566 Wild Type fmr1 KO fmr1 KO mice show an increase in testis weight, as do human Fragile X Syndrome patients NNZ-2566 has no significant effect in Wild Type mice After NNZ-2566 administration to fmr1 KO mice, testis weight is not significantly different from controls
Ras-Mek-Erk Pathway Vehicle NNZ-2566 Vehicle NNZ-2566 N = 4 per Group Vehicle NNZ-2566 Vehicle NNZ-2566 Overall levels of ERK are unchanged in the brain in fmr1 KO mice However, activation of ERK, as indexed by levels of phosphorylated ERK, is increased This activation is normalized by treatment with NNZ-2566 NNZ-2566 may also increase total ERK levels in fmr1 KO mice
Akt-Pathway pakt Vehicle vehicle NNZ-2566 NNZ WT KO WT KO AKT N = 4 per Group Vehicle NNZ-2566 Activation of Akt, as indexed by levels of phosphorylation, is increased This activation is normalized by treatment with NNZ-2566
Effects in Rett Syndrome model 29
Pharmacological Reversal of Phenotype IGF-1[1-3] reverses phenotype MECP2 mouse Tropea et al. 2009 30
NNZ-2566 Rett Syndrome / Fragile X Syndrome Recruitment into Phase II clinical trial in Rett Syndrome is underway 48 subjects; 2 doses plus placebo; 14 day follow-up Adolescents and adults Subjects will have baseline assessments of EEG, cardiac and respiratory rhythms, and functional status Focus on safety and signals of efficacy Large market opportunity Significant unmet need; no marketed therapeutic Symptoms amenable to treatment Potential for disease modification? Potential gateway to autism Fragile X Syndrome study planning underway 31
Summary NNZ-2566 shows efficacy in pre-clinical models of: Traumatic brain injury Fragile X Syndrome Rett Syndrome Efficacy accompanied by: Decrease in microglial activation Decrease in production of inflammatory cytokines Normalization of Akt and ERK activation profiles Clinical studies now underway 32