Francesco G. Salituro

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.

Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr–/y mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ‐aminobutyric acidergic (GABAergic) transmission provided by peri‐ and extrasynaptic GABA type A (GABAA) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1–/y knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1–/y KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE‐872, based on endogenously active neurosteroids. This study shows that SGE‐872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 μM), SGE‐872 is selective for tonic, extrasynaptic α4β3δ‐containing GABAA receptors over typical synaptic α1β2γ2 receptors. We further find that SGE‐872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1–/y KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE‐872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

Chapter Three – Natural and Synthetic Neuroactive Steroid Modulators of GABAA and NMDA Receptors

Abstract Neuroactive steroids (NASs) are a family of steroid-based compounds of both natural and synthetic origin, which have been shown to impact central nervous system function through allosteric modulation of the GABA(γ-aminobutyric acid)A receptor and the NMDA ( N -methyl- d -aspartic acid) class of glutamate receptors. Respectively, these are two of the major inhibitory and excitatory neurotransmitter receptor families involved in synaptic transmission in the brain. Research over recent years has demonstrated that these receptors can be either positively (PAM) or negatively (NAM) allosterically modulated by NAS compounds, an action mediated through binding to allosteric sites. The focus of this report is to briefly review endogenous and synthetic NASs that target GABA A and NMDA receptors.

Anticonvulsant profile of the neuroactive steroid, SGE-516, in animal models

Despite the availability of multiple antiepileptic drugs (AED), failure to adequately control seizures is a challenge for approximately one third of epilepsy patients, and new therapies with a differentiated mechanism of action are needed. The neuroactive steroid, SGE-516, is a positive allosteric modulator of both gamma- and delta-containing GABAA receptors. This broad GABAA receptor activity differentiates neuroactive steroids like SGE-516 from benzodiazepines, a class of anticonvulsants which have been shown in vitro to selectively target gamma-subunit containing GABAA receptors. As a neuroactive steroid, SGE-516 has pharmacokinetic properties that are intended to allow for chronic oral dosing. We investigated the anticonvulsant activity of SGE-516 across numerous in vitro and in vivo models of seizure activity. SGE-516 dose-dependently reduced neuronal firing rates and epileptiform activity in vitro. In mice, SGE-516 protected against acute seizures in the PTZ-induced chemo-convulsant seizure model and the 6Hz psychomotor seizure model. In addition, SGE-516 demonstrated anticonvulsant activity in the mouse corneal kindling model. These data suggest that SGE-516 may have potential for development as a novel oral AED for the treatment of refractory seizures.

Dual p38/JNK mitogen activated protein kinase inhibitors prevent ozone-induced airway hyperreactivity in guinea pigs.

Ozone exposure causes airway hyperreactivity and increases hospitalizations resulting from pulmonary complications. Ozone reacts with the epithelial lining fluid and airway epithelium to produce reactive oxygen species and lipid peroxidation products, which then activate cell signaling pathways, including the mitogen activated protein kinase (MAPK) pathway. Both p38 and c-Jun NH2 terminal kinase (JNK) are MAPK family members that are activated by cellular stress and inflammation. To test the contribution of both p38 and JNK MAPK to ozone-induced airway hyperreactivity, guinea pigs were pretreated with dual p38 and JNK MAPK inhibitors (30 mg/kg, ip) 60 minutes before exposure to 2 ppm ozone or filtered air for 4 hours. One day later airway reactivity was measured in anesthetized animals. Ozone caused airway hyperreactivity one day post-exposure, and blocking p38 and JNK MAPK completely prevented ozone-induced airway hyperreactivity. Blocking p38 and JNK MAPK also suppressed parasympathetic nerve activity in air exposed animals, suggesting p38 and JNK MAPK contribute to acetylcholine release by airway parasympathetic nerves. Ozone inhibited neuronal M2 muscarinic receptors and blocking both p38 and JNK prevented M2 receptor dysfunction. Neutrophil influx into bronchoalveolar lavage was not affected by MAPK inhibitors. Thus p38 and JNK MAPK mediate ozone-induced airway hyperreactivity through multiple mechanisms including prevention of neuronal M2 receptor dysfunction.

Breakthroughs in neuroactive steroid drug discovery

Endogenous and synthetic neuroactive steroids (NASs) or neurosteroids are effective modulators of multiple signaling pathways including receptors for the γ-aminobutyric acid A (GABAA) and glutamate, in particular N-methyl-d-aspartate (NMDA). These receptors are the major inhibitory and excitatory neurotransmitters in the central nervous system (CNS), and there is growing evidence suggesting that dysregulation of neurosteroid production plays a role in numerous neurological disorders. The significant unmet medical need for treatment of CNS disorders has increased the interest for these types of compounds. In this review, we highlight recent progress in the clinical development of NAS drug candidates, in addition to preclinical breakthroughs in the identification of novel NASs, mainly for GABAA and NMDA receptor modulation.