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Novel brain permeant mTORC1/2 inhibitors are as efficacious as rapamycin or everolimus in mouse models of acquired partial epilepsy and tuberous sclerosis complex

Theilmann, W,Gericke,B,Schidlitzki, A, Anjum, S,Borsdorf, S, Harries, T, Roberds, S.L., Aguiar, D,Brunner, D, Leiser, S,Song, D, Fabbro, D,Hillmann, P, Wymann, M,Loscher, W. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2020.108297.
Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative (“rapalog”) everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of two novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors, PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1GFAP CKO mice, a well-characterized mouse model of epilepsy in TSC. During prolonged treatment of IHK mice with rapamycin, everolimus, PQR620, PQR626, or PQR530; only PQR620 exerted a transient antiseizure effect on SRS, at well tolerated doses whereas the other compounds were ineffective. In contrast, all of the examined compounds markedly suppressed SRS in Tsc1GFAP CKO mice during chronic treatment at well tolerated doses. Thus, against our expectation, no clear differences in antiseizure efficacy were found across the three classes of mTOR inhibitors examined in mouse models of genetic and acquired epilepsies. The main advantage of the novel 1,3,5-triazine derivatives is their excellent tolerability compared to rapalogs, which would favor their development as new therapies for TORopathies such as TSC.

Mouse model systems of autism spectrum disorder: Replicability and informatics signature

Kabitzke, P, Morales, D, He, D, Cox, K, Sutphen, J, Thiede, L, Sabath, E, Hanania, T, Biemans, B, Brunner, D. Genes, Brain and Behavior. 2020;e12676.
Phenotyping mouse model systems of human disease has proven to be a difficult task, with frequent poor inter‐ and intra‐laboratory replicability, particularly in behavioral domains such as social and cognitive function. However, establishing robust animal model systems with strong construct validity is of fundamental importance as they are central tools for understanding disease pathophysiology and developing therapeutics. To complete our studies of mouse model systems relevant to autism spectrum disorder (ASD), we present a replication of the main findings from our two published studies of five genetic mouse model systems of ASD. To assess the intra‐laboratory robustness of previous results, we chose the two model systems that showed the greatest phenotypic differences, the Shank3/F and Cntnap2, and repeated assessments of general health, activity and social behavior. We additionally explored all five model systems in the same framework, comparing all results obtained in this three‐yearlong effort using informatics techniques to assess commonalities and differences. Our results showed high intra‐laboratory replicability of results, even for those with effect sizes that were not particularly large, suggesting that discrepancies in the literature may be dependent on subtle but pivotal differences in testing conditions, housing enrichment, or background strains and less so on the variability of the behavioral phenotypes. The overall informatics analysis suggests that in our behavioral assays we can separate the set of tested mouse model system into two main classes that in some aspects lie on opposite ends of the behavioral spectrum, supporting the view that autism is not a unitary concept.

ANAVEX®2-73 (blarcamesine), a Sigma-1 receptor agonist, ameliorates neurologic impairments in a mouse model of Rett syndrome

Kaufmann W E, Sprouse J, Rebowe N, Hanania T, Klamer D, Missling C U Pharmacol Biochem Behav. 2019, December 187:172796. doi: 10.1016/j.pbb.2019.172796.
Rett syndrome (RTT) is a severe neurodevelopmental disorder that is associated in most cases with mutations in the transcriptional regulator MECP2. At present, there are no effective treatments for the disorder. Despite recent advances in RTT genetics and neurobiology, most drug development programs have focused on compounds targeting the IGF-1 pathway and no pivotal trial has been completed as yet. Thus, testing novel drugs that can ameliorate RTT's clinical manifestations is a high priority. ANAVEX2-73 (blarcamesine) is a Sigma-1 receptor agonist and muscarinic receptor modulator with a strong safety record and preliminary evidence of efficacy in patients with Alzheimer's disease. Its role in calcium homeostasis and mitochondrial function, cellular functions that underlie pathological processes and compensatory mechanisms in RTT, makes blarcamesine an intriguing drug candidate for this disorder. Mice deficient in MeCP2 have a range of physiological and neurological abnormalities that mimic the human syndrome. We tested blarcamesine in female heterozygous mice carrying one null allele of Mecp2 (HET) using a two-tier approach, with age-appropriate tests. Administration of the drug to younger and older adult mice resulted in improvement in multiple motor, sensory, and autonomic phenotypes of relevance to RTT. The latter included motor coordination and balance, acoustic and visual responses, hindlimb clasping, and apnea in expiration. In line with previous animal and human studies, blarcamesine also showed a good safety profile in this mouse model of RTT. Clinical studies in RTT with blarcamesine are ongoing.

SEP-363856, a Novel Psychotropic Agent with a Unique, Non-D2 Receptor Mechanism of Actions

Dedic N, Jones P.G, Hopkins S.C, Lew R, Shao L, Campbell J. E, Spear K. L, Large T. H, Campbell U. C, Hanania T, Leahy E, Koblan K Journal of Pharmacology and Experimental Therapeutics. August 1, 2019 jpet.119.260281.
For the past 50 years, the clinical efficacy of antipsychotic medications has relied on blockade of dopamine D2 receptors. Drug development of non-D2 compounds, seeking to avoid the limiting side effects of dopamine receptor blockade, has failed to date to yield new medicines for patients. In this work, we report the discovery of SEP-363856 (SEP-856), a novel psychotropic agent with a unique mechanism of action. SEP-856 was discovered in a medicinal chemistry effort utilizing a high throughput, high content, mouse-behavior phenotyping platform, in combination with in vitro screening, aimed at developing non-D2 (anti-target) compounds that could nevertheless retain efficacy across multiple animal models sensitive to D2-based pharmacological mechanisms. SEP-856 demonstrated broad efficacy in putative rodent models relating to aspects of schizophrenia, including phencyclidine (PCP)-induced hyperactivity, prepulse inhibition, and PCP-induced deficits in social interaction. In addition to its favorable pharmacokinetic properties, lack of D2 receptor occupancy, and the absence of catalepsy, SEP-856’s broad profile was further highlighted by its robust suppression of rapid eye movement sleep in rats.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington’s disease

Zeitler B, Froelich S, Marlen K, Shivak D.A, Yu Q, Li D, Pearl J.R, Miller J.C, Zhang L, Paschon D.E, Hinkley S.J, Ankoudinova I, Lam S, Guschin D, Kopan L, Cherone J.M, Nguyen B.H.O, Qiao G, Ataei Y, Mendel M.C, Amora R, Surosky R, Laganiere J, Vu B.J , Narayanan A, Sedaghat Y, Tillack K, Thiede C, Gärtner A, Kwak S, Bard J, Mrzljak L, Park L, Heikkinen T, Lehtimäki K.K , Svedberg M.M, Häggkvist J, Tari L, Tóth M, Varrone A, Halldin C, Kudwa A.E, Ramboz S, Day M, Kondapalli J, Surmeier D.J, Urnov F.D, Gregory P.D, Rebar E.J, Sanjuán I.M, Zhang H.S Nature Medicine. July 25, 2019. Vol. 25, 1131–1142.
Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Validated Phenotypic Approach to Neuropsychiatric Drug Discovery

Leahy E Drug Development & Delivery March 2019 Vol 19 No 2: 46-49.
At the recent American College of Neuropsychopharmacology (ACNP) meeting, Sunovion Pharmaceuticals and PsychoGenics Inc. announced positive Phase 2 results for SEP-363856, a novel, first-in-class treatment that has the potential to offer patients with schizophrenia the chance to live a near-normal life. Available treatment options fail to treat all schizophrenia symptoms, and their side effects result in non-compliance and relapse. SEP-363856, by contrast, shows robust effects across a broad range of disabling symptoms, including positive, negative, depressive, and general psychopathology symptoms, with a safety profile similar to placebo. Discovered via SmartCube®, PsychoGenics’ target-agnostic platform, SEP-363856 does not interact with the dopamine D2 or other neuroreceptors thought to mediate the effects of currently available antipsychotic agents. The SmartCube platform thus represents a novel approach to discovering the next generation of breakthrough treatments for schizophrenia and other neuropsychiatric disorders.

Biomarker Analysis of Orally Dosed, Dual Active, Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitor, AQU-118, in the Spinal Nerve Ligation (SNL) Rat Model of Neuropathic Pain

Kwan M Y, Choo A, Hanania T, Ghavami A, Beltran J, Shea J, Barboza A, Hu A, Fowler M, Rao N V, Sucholeiki I Int J Mol Sci. 2019 Feb; 20(4): 811. Published online 2019 Feb 14. doi: 10.3390/ijms20040811.
There is an unmet medical need for the development of non-addicting pain therapeutics with enhanced efficacy and tolerability. The current study examined the effects of AQU-118, an orally active inhibitor of metalloproteinase-2 (MMP-2) and MMP-9, in the spinal nerve ligation (SNL) rat model of neuropathic pain. Mechanical allodynia and the levels of various biomarkers were examined within the dorsal root ganglion (DRG) before and after oral dosing with AQU-118. The rats that received the SNL surgery exhibited significant mechanical allodynia as compared to sham controls. Animals received either vehicle, positive control (gabapentin), or AQU-118. After SNL surgery, the dorsal root ganglion (DRG) of those rats dosed with vehicle had elevated messenger RNA (mRNA) expression levels for MMP-2, IL1-β & IL-6 and elevated protein levels for caspase-3 while exhibiting decreased protein levels for myelin basic protein (MBP) & active IL-β as compared to sham controls. Rats orally dosed with AQU-118 exhibited significantly reduced mechanical allodynia and decreased levels of caspase-3 in the DRG as compared to vehicle controls. Results demonstrate that oral dosing with the dual active, MMP-2/-9 inhibitor, AQU-118, attenuated mechanical allodynia while at the same time significantly reduced the levels of caspase-3 in the DRG.

Myelinated axons fail to develop properly in a genetically authentic mouse model of Charcot-Marie-Tooth disease type 2E

Lancaster, Li J, Hanania T, Liem R, Scheideler M. A, Scherer S. Experimental Neurology 2018 Oct;308:13-25. doi: 10.1016/j.expneurol.2018.06.010. Epub 2018 Jun 22.
We have analyzed a mouse model of Charcot-Marie-Tooth disease 2E (CMT2E) harboring a heterozygous p.Asn98Ser (p.N98S) Nefl mutation, whose human counterpart results in a severe, early-onset neuropathy. Behavioral, electrophysiological, and pathological analyses were done on separate cohorts of NeflN98S/+ mutant mice and their wild type Nefl+/+ littermates between 8 and 48 weeks of age. The motor performance of NeflN98S/+ mice, as evidenced by altered balance and gait measures, was impaired at every age examined (from 6 to 25 weeks of age). At all times examined, myelinated axons were smaller and contained markedly fewer neurofilaments in NeflN98S/+ mice, in all examined aspects of the PNS, from the nerve roots to the distal ends of the sciatic and caudal nerves. Similarly, the myelinated axons in the various tracts of the spinal cord and in the optic nerves were smaller and contained fewer neurofilaments in mutant mice. The myelinated axons in both the PNS and the CNS of mutant mice had relatively thicker myelin sheaths. The amplitude and the nerve conduction velocity of the caudal nerves were reduced in proportion with the diminished sizes of myelinated axons. Conspicuous aggregations of neurofilaments were only seen in primary sensory and motor neurons, and were largely confined to the cell bodies and proximal axons. There was evidence of axonal degeneration and regeneration of myelinated axons, mostly in distal nerves. In summary, the p.N98S mutation causes a profound reduction of neurofilaments in the myelinated axons of the PNS and CNS, resulting in substantially reduced axonal diameters, particularly of large myelinated axons, and distal axon loss in the PNS.

Differences in Synaptic Dysfunction Between rTg4510 and APP/PS1 Mouse Models of Alzheimer’s Disease

Gelman S, Palma J, Tombaugh G, Ghavami A Journal of Alzheimer's Disease, vol. 61, no. 1, pp. 195-208, 2018 DOI: 10.3233/JAD-170457
Genetically modified mice have provided insights into the progression and pathology of Alzheimer’s disease (AD). Here, we have examined two mouse models of AD: the rTg4510 mouse, which overexpresses mutant human Tau gene, and the APP/PS1 mouse, which overexpresses mutant human genes for amyloid precursor protein and presenilin 1. Both models exhibit deficits in hippocampal function, but comparative analyses of these deficits are sparse. We used extracellular field potential recordings in hippocampal slices to study basal synaptic transmission (BST), paired-pulse facilitation (PPF), and long-term potentiation (LTP) at the Schaffer collateral-CA1 pyramidal cell synapses in both models. We found that 6-7, but not 2-3-month-old rTg4510 mice exhibited reduced pre-synaptic activation (fiber volley (FV) amplitude, ∼50%) and field excitatory post-synaptic potential (fEPSP) slope (∼40%) compared to wild-type controls. In contrast to previous reports, BST, when controlled for FV amplitude, was not altered in rTg4510. APP/PS1 mice (2-3 mo and 8-10 mo) had unchanged FV amplitude compared to wild-type controls, while fEPSP slope was reduced by ∼34% in older mice, indicating a deficit in BST. PPF was unchanged in 8–10-month-old APP/PS1 mice, but was reduced in 6-7-month-old rTg4510 mice. LTP was reduced only in older rTg4510 and APP/PS1 mice. Our data suggest that BST deficits appear earlier in APP/PS1 than in rTg4510, which exhibited no BST deficits at the ages tested. However, FV and synaptic plasticity deficits developed earlier in rTg4510. These findings highlight fundamental differences in the progression of synaptic pathology in two genetically distinct models of AD.