Christine M. Solinsky

Frontiers in therapeutic development of allopregnanolone for Alzheimer’s disease and other neurological disorders

Allopregnanolone (Allo), a neurosteroid, has emerged as a promising promoter of endogenous regeneration in brain. In a mouse model of Alzheimer’s disease, Allo induced neurogenesis, oligodendrogenesis, white matter generation and cholesterol homeostasis while simultaneously reducing β-amyloid and neuroinflammatory burden. Allo activates signaling pathways and gene expression required for regeneration of neural stem cells and their differentiation into neurons. In parallel, Allo activates systems to sustain cholesterol homeostasis and reduce β-amyloid generation. To advance Allo into studies for chronic human neurological conditions, we examined translational and clinical parameters: dose, regimen, route, formulation, outcome measures, and safety regulations. A treatment regimen of once per week at sub-sedative doses of Allo was optimal for regeneration and reduction in Alzheimer’s pathology. This regimen had a high safety profile following chronic exposure in aged normal and Alzheimer’s mice. Formulation of Allo for multiple routes of administration has been developed for both preclinical and clinical testing. Preclinical evidence for therapeutic efficacy of Allo spans multiple neurological diseases including Alzheimer’s, Parkinson’s, multiple sclerosis, Niemann-Pick, diabetic neuropathy, status epilepticus, and traumatic brain injury. To successfully translate Allo as a therapeutic for multiple neurological disorders, it will be necessary to tailor dose and regimen to the targeted therapeutic mechanisms and disease etiology. Treatment paradigms conducted in accelerated disease models in young animals have a low probability of successful translation to chronic diseases in adult and aged humans. Gender, genetic risks, stage and burden of disease are critical determinants of efficacy. This review focuses on recent advances in development of Allo for Alzheimer’s disease (AD) that have the potential to accelerate therapeutic translation for multiple unmet neurological needs.

Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for alzheimer's disease

To develop allopregnanolone as a therapeutic for Alzheimer’s disease, we investigated multiple formulations and routes of administration in translationally relevant animal models of both sexes. Subcutaneous, topical (transdermal and intranasal), intramuscular, and intravenous allopregnanolone were bolus-administered. Pharmacokinetic analyses of intravenous allopregnanolone in rabbit and mouse indicated that peak plasma and brain levels (3-fold brain/plasma ratios) at 5min were sufficient to activate neuroregenerative responses at sub-sedative doses. Slow-release subcutaneous suspension of allopregnanolone displayed 5-fold brain/plasma ratio at Cmax at 30min. At therapeutic doses by either subcutaneous or intravenous routes, allopregnanolone mouse plasma levels ranged between 34-51ng/ml by 30min, comparable to published endogenous human level in the third trimester of pregnancy. Exposure to subcutaneous, topical, intramuscular, and intravenous allopregnanolone, at safe and tolerable doses, increased hippocampal markers of neurogenesis including BrdU and PCNA in young 3xTgAD and aged wildtype mice. Intravenous allopregnanolone transiently and robustly phosphorylated CREB within 5min and increased levels of neuronal differentiation transcription factor NeuroD within 4h. Neurogenic efficacy was achieved with allopregnanolone brain exposure of 300-500hr*ng/g. Formulations were tested to determine the no observable adverse effect level (NOAEL) and maximally tolerated doses (MTD) in male and female rats by sedation behavior time course. Sex differences were apparent, males exhibited ≥40% more sedation time compared to females. Allopregnanolone formulated in sulfobutyl-ether-beta-cyclodextrin at optimized complexation ratio maximized allopregnanolone delivery and neurogenic efficacy. To establish the NOAEL and MTD for Allo-induced sedation using a once-per-week intravenous regenerative treatment regimen: In female rats the NOAEL was 0.5mg/kg and MTD 2mg/kg. The predicted MTD in human female is 0.37mg/kg. In male rats the NOAEL and MTD were less than those determined for female. Outcomes of these PK/PD studies predict a safe and efficacious dose range for initial clinical trials of allopregnanolone for Alzheimer’s disease. These findings have translational relevance to multiple neurodegenerative conditions.

Development of a unified clinical trial database for Alzheimer's disease.

Data obtained in completed Alzheimers disease (AD) clinical trials can inform decision making for future trials. Recognizing the importance of sharing these data, the Coalition Against Major Diseases created an Online Data Repository for AD (CODR‐AD) with the aim of supporting accelerated drug development. The aim of this study was to build an open access, standardized database from control arm data collected across many clinical trials.

DEVELOPMENT OF AN IPSC-BASED BIOMARKER STRATEGY TO IDENTIFY NEUROREGENERATIVE RESPONDERS TO ALLOPREGNANOLONE

Madhav Thambisetty, Cristina Legido-Quigley, King’s College London, London, United Kingdom; Kings College London, London, United Kingdom; Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, United Kingdom; NIHR Biomedical Research Centre for Mental Health and Biomedical Research Unit for Dementia at South London and Maudsley NHS Foundation, London, United Kingdom; Johns Hopkins University School of Medicine, Baltimore, MD, USA; Duke University, Durham, NC, USA; National Institute on Aging, Baltimore, MD, USA. Contact e-mail: stuart.snowden@kcl.ac.uk

COMPARATIVE PHARMACOKINETIC ASSESSMENT OF ALLOPREGNANOLONE TO DEVELOP A REGENERATIVE THERAPEUTIC FOR MILD COGNITIVE IMPAIRMENT AND EARLY ALZHEIMER’S DISEASE

Background: Currently in Phase 1b, Allopregnanolone (Allo) is in development as the first regenerative therapeutic for Alzheimer’s disease (AD). Pharmacokinetic (PK) outcomes in animals estimate the maximally tolerated dose (MTD) and no observed adverse effect level (NOAEL). Dose-escalating clinical PK studies (placebocontrolled) are underway to evaluate safe doses of Allo in men and women age 55+ with MCI/earlyAD (NCT02221622). Methods: Allo plasma concentrations (LC-MS/MS) were compared in: 1) 5-month 3xTgAD or 15-month nonTg male mice treated acutely via s.c., i.m., t.d., i.n., i.v. 2) Adult male and female Sprague Dawley rats at doses from 1-8 mg/kg i.m. 3) Adult New Zealand White female rabbits at 3mg/kg t.d. and 5mg/kg i.v. 4) Adult male and female Beagles at doses from 2.4-6 mg/kg i.m. and i.v 5) Men and women age >55years with MMSE >20 diagnosed with MCI/earlyAD dosed by 30min. i.v. infusion. Results: PK profiles frommultiple species were compared acrossMTD,NOAEL,Cmax, Tmax, AUC and half-life. Across species, Allo plasma levels w1000ng/ml elicited gait ataxia; abovew1500ng/ml elicited moderate to deep sedation within 5-15minutes. We estimate 3-fold brain/plasma ratios across multiple routes and formulations, useful for understanding MTD limits. Sedation effects at high-doses were more enhanced in males than females (mg/kg) despite similar plasma concentrations. Cmax and AUClast increased with ascending dose levels, approximately proportional to dose. Cmaxplasma 33ng/ml; brain 160ng/g elicited efficacy in ADmouse models indicating that, relative to MTD, moderate plasma Allo is sufficient. Half-life values were <1hr for most routes and formulations. Conclusions:Acute administration of Allo in several species allowed us to compare PKparameters to assess safety and establish a scalable dose range predicted to be efficacious. Previously we showed that Allo transiently and robustly phosphorylated CREB within 5min and increased levels of NeuroD within 4h (Irwin et al., 2015) at mild to moderate doses. Allo as a regenerative therapeutic is based on well-defined mechanistic targets for neurogenesis and disease modification, dosing, formulation, route of administration, and the appropriate treatment regimen necessary to advance to Phase2 for MCI/earlyAD. Research supported by NIA U01 AG031115; NIA U01 AG047222; UF1 AG046148 and ADDF to RDB.

Allopregnanolone chronic exposure using regenerative treatment regimen: Preclinical ind enabling toxicology indicating safety

Background: Diet and nutrition may play an important role in neurocognitive health. Whether and how effective can single nutrients, or diet patterns be protective against neurocognitive decline, remains controversial. In this paper we review data from cohort studies, case-control studies and RCT relating either signal nutrients or dietary pattern to the risk of cognitive decline and dementia. We focus on the following six groups: caloric restriction, alcohol, vitamin B, antioxidants, fatty acids, and dietary patterns. Methods:We systematically reviewed selected modifiable dietary factors including calorie, alcohol, vitamin B, antioxidants, fatty acids, and dietary patterns that were studied in relation to neurocognitive health, including incident dementia. We searched MEDLINE, EMBASE and SCOPUS for published literature, excluding cross-sectional studies and laboratory trials. Analysis compared study finding consistency across factors, study designs e.g. population size, dosage of supplementation, age group and study-level characteristics. Results: In total, 90 studies were retrieved for systematic review. Research findings are mostly inconsistent even for those mostly studied compound such as vitamin B and antioxidant. Studies indicating daily energy deficit not significantly related to change in cognitive function except for DASH diet combined with a weight reduction program, however limited due to small sample size. A few RCT and cohort studies suggested no significant association between unsaturated fatty acids intake and incident dementia while others provide evidence of the opposite. Most studies found low to moderate alcohol intake lower risk of dementia. Dietary Patterns which is characterized by higher intake of fruits, vegetables, fish, nuts and legumes, and lower intake of meats and butter seemed to be associated with reduced risk of dementia. Conclusions: Conflicting data is found due to methodology issues. Further studies need focus on specific age group, adequate sample size and follow up years, subjects whose diet is assessed and monitored at an earlier stage and adequate dosage of supplementation on nutrient of interest.

Early-stage development of ips-based biomarkers to identify patient population with a regenerative profile responsive to allopregnanolone

Background: ALL1-fused from chromosome 1q (AF1q), originally considered as an oncogenic factor, has been implicated in the pathogenesis of neurodegeneration.AF1q is highly expressed during neurodevelopment, but its specific functions and molecular mechanisms in neural system remained elusive. Methods:Luciferase reporter assay, Co-IP, Edu cell proliferation assay, Western Blot, Site-directed mutagenesis. Results:Our study here demonstrated that AF1q facilitated neural stem cell proliferation. Since previous studies have shown WNT signaling being involved in neural stem cell proliferation, we examined whether AF1q could induce cell proliferation by activating Wnt signaling pathway. Reporter assay showed AF1Q could activate WNT signaling. And co-immunoprecipitation (CO-IP) analysis demonstrated that AF1q bound specifically to T-cell factor/lymphoid enhancer binding factor-7 (TCF/LEF7), which stabilized TCF7 and facilitated TCF7 translocation into nucleus. Additionally, we identified the amino acid 11-20 on AF1Q is sufficient for the binding of TCF7. Furthermore, the phosphorylation of Serine 11 on AF1Q is required for the binding of TCF7. The AF1Q-S11F mutant decreased the activation of WNT signaling and was unable to induce cell proliferation. Conclusions: Our study here identified AF1Q as an important factor in neurodevelopment by interacting with TCF7 and regulating WNT signaling pathway.