JPET Fast Forward. Published on December 14, 2018 as DOI: 10.1124/jpet.118.252155 This article has not been copyedited and formatted. The final version may differ from this version. JPET #252155 TITLE PAGE Intravenously Administered Ganaxolone Blocks Diazepam- Resistant Lithium- Pilocarpine-Induced Status Epilepticus in Rats. Comparison with Allopregnanolone Michael S. Saporito1, John A. Gruner2, Amy DiCamillo2, Richard Hinchliffe2, Downloaded from Melissa Barker-Haliski3, H. Steven White3 jpet.aspetjournals.org 1 Marinus Pharmaceuticals Inc. Radnor, PA 2 Melior Discovery Inc. Exton, PA 3 Department of Pharmacy, School of Pharmacy, University of Washington, Seattle, at ASPET Journals on September 30, 2021 WA 1 JPET Fast Forward. Published on December 14, 2018 as DOI: 10.1124/jpet.118.252155 This article has not been copyedited and formatted. The final version may differ from this version. JPET #252155 RUNNING TITLE PAGE Running Title: Ganaxolone blocks diazepam resistant status epilepticus *Address correspondence to: Downloaded from Michael S. Saporito, Ph.D. Marinus Pharmaceuticals, Inc. 170 N. Radnor Chester Rd. Suite 250 jpet.aspetjournals.org Radnor, PA 19087 Email: [email protected] at ASPET Journals on September 30, 2021 Phone: 484-801-4670 Manuscript statistics: Total text pages: 30 Tables: 4 Figures: 7 Supplemental Figures: 1 Abstract: 228 words Introduction, 701 words Discussion, 1490 words Topic Category: Neuropharmacology 2 JPET Fast Forward. Published on December 14, 2018 as DOI: 10.1124/jpet.118.252155 This article has not been copyedited and formatted. The final version may differ from this version. JPET #252155 Abbreviations: ALLO: allopregnanolone GNX: ganaxolone SE: status epilepticus CSE: Convulsive status epilepticus EEG: electroencephalographic Downloaded from JVC: jugular vein catheter FFT: Fast Fourier Transform jpet.aspetjournals.org LORR: loss of righting reflex AED: Antiepileptic Drug at ASPET Journals on September 30, 2021 DZP: diazepam IM: intramuscular IV: intravenous PK: Pharmacokinetic PD: Pharmacodynamic 3 JPET Fast Forward. Published on December 14, 2018 as DOI: 10.1124/jpet.118.252155 This article has not been copyedited and formatted. The final version may differ from this version. JPET #252155 Abstract Ganaxolone (GNX) is the 3β-methylated synthetic analog of the naturally occurring neurosteroid, allopregnanolone (ALLO). GNX is effective in a broad range of epilepsy and behavioral animal models and is currently in clinical trials designed to assess its anticonvulsant and antidepressant activities. The current studies were Downloaded from designed to broaden the anticonvulsant profile of GNX by evaluating its potential anticonvulsant activities following intravenous (IV) administration in treatment resistant models of status epilepticus (SE), to establish a pharmacokinetic jpet.aspetjournals.org (PK)/pharmacodynamics (PD) relationship, and to compare its PK and anticonvulsant activities to ALLO. In PK studies, GNX had higher exposure levels, a at ASPET Journals on September 30, 2021 longer half-life, slower clearance, and higher brain penetrance than ALLO. Both GNX and ALLO produced a sedating response as characterized by loss of righting reflex, but neither compound produced a full anesthetic response as animals still responded to painful stimuli. Consistent with their respective PK properties, the sedative effect of GNX was longer than that of ALLO. Unlike other non-anesthetizing anticonvulsant agents indicated for SE, both GNX and ALLO produced anticonvulsant activity in models of pharmacoresistant SE with administration delay times of up to one hour after seizure onset. Again, consistent with their respective PK properties, GNX produced a significantly longer anticonvulsant response. These studies show that GNX exhibited improved pharmacological characteristics versus other agents used as treatments for SE and position GNX as a uniquely acting treatment for this indication. 4 JPET Fast Forward. Published on December 14, 2018 as DOI: 10.1124/jpet.118.252155 This article has not been copyedited and formatted. The final version may differ from this version. JPET #252155 Introduction Naturally occurring neurosteroids, such as allopregnanolone (ALLO), elicit a broad range of anticonvulsant and psychotherapeutic responses in experimental animal models and are currently being evaluated for these activities in human clinical trials (Frye, 1995; Kanes et al., 2017; Kokate et al., 1994; Rosenthal et al., 2017). Neurosteroids elicit their anticonvulsant activities through positive allosteric modulation of endogenous GABAA receptors located in the CNS (Belelli et al., 2006; Downloaded from Belelli and Lambert, 2005). Both neurosteroids and benzodiazepines positively modulate synaptically located GABAA receptors comprised of α and γ subunits jpet.aspetjournals.org (Campo-Soria et al., 2006). However, neurosteroids act via a distinct binding site on the GABAA receptor and, unlike benzodiazepines, also modulate extrasynaptic GABAA receptors that are comprised of α and δ subunits (Akk et al., 2004; Belelli et at ASPET Journals on September 30, 2021 al., 2006; Belelli and Lambert, 2005; Campo-Soria et al., 2006; Sigel and Steinmann, 2012). This distinctive receptor selectivity confers a unique pharmacological profile to neurosteroids. However, the utility of naturally occurring neurosteroids as therapeutics are limited by their pharmacokinetic liabilities including lack of oral bioavailability and metabolic stability (Carter et al., 1997; Frye, 1995; Kokate et al., 1994; Martinez Botella et al., 2015). Ganaxolone (GNX; CCD-1042; 3β-Methyl-3α-ol-5α-pregnan-20-one; 3α-Hydroxy-3β- methyl-5α-pregnan-20-one) differs from naturally occurring ALLO by addition of a methyl group at the 3-position (Carter et al., 1997). The 3β-methylation prevents back conversion to the hormonally active 3-keto derivative, eliminates affinity to the nuclear hormone progesterone receptor, and confers metabolic stability and oral 5 JPET Fast Forward. Published on December 14, 2018 as DOI: 10.1124/jpet.118.252155 This article has not been copyedited and formatted. The final version may differ from this version. JPET #252155 bioavailability in experimental animals and humans (Carter et al., 1997; Nohria and Giller, 2007). Moreover, this chemical modification does not meaningfully modify the potency, efficacy, or selectivity of GNX to GABAA receptors (Carter et al., 1997; Nik et al., 2017). GNX is effective in a broad range of animal models of epilepsy and behavioral disorders, but exhibits important pharmacological differences from the Downloaded from benzodiazepine class of GABAA receptor modulators (Carter et al., 1997; Pinna and Rasmusson, 2014; Reddy and Rogawski, 2000, 2010; Yum et al., 2014). Unlike benzodiazepines, repeated GNX administration does not induce tolerance to the jpet.aspetjournals.org anticonvulsant response and there is greater separation between anticonvulsant and sedating doses (Gasior et al., 1997; Gasior et al., 2000; Mares and Stehlikova, 2010). On the basis of these distinctive pharmacological properties and broad at ASPET Journals on September 30, 2021 preclinical efficacy, GNX is currently being evaluated for behavioral effects and anticonvulsant activities in clinical studies (Younus and Reddy, 2018). Status epilepticus (SE) is an especially severe and life-threatening condition that frequently occurs in patients with epilepsy, as well as individuals without a history of epilepsy. Patients in SE almost always require treatment with parenterally (typically IV) administered drugs (Glauser et al., 2016). In clinical settings, the first- line treatment for control of SE is IV -administration of benzodiazepines (Glauser et al., 2016). In patients for whom benzodiazepine treatment fails, the guidelines call for sequential treatment with standard anticonvulsant drugs such as phenytoin, valproic acid, phenobarbital and/or levetiracetam. If SE remains uncontrolled, treatment with general anesthetics such as pentobarbital or propofol is initiated 6 JPET Fast Forward. Published on December 14, 2018 as DOI: 10.1124/jpet.118.252155 This article has not been copyedited and formatted. The final version may differ from this version. JPET #252155 (Glauser et al., 2016). Patients with SE become progressively refractory to treatment over time from onset, and up to 30% of patients with SE cannot be successfully treated and die within 30 days (Al-Mufti and Claassen, 2014; Trinka et al., 2015). Thus, there is a clear unmet medical need for additional therapeutics effective against treatment resistant forms of SE. The lithium-pilocarpine rodent model of SE is a clinically translatable model of SE Downloaded from (Curia et al., 2008; Jones et al., 2002). Both the rodent model and clinical SE exhibit convulsive and electroencephalographic (EEG) seizures, mortality and, in subjects that survive, cognitive deficits and neuronal degeneration(Lehmkuhle et al., 2009; jpet.aspetjournals.org Tang et al., 2011; White et al., 2012). Moreover, animal subjects exhibit similar response profiles to treatments that are effective in clinical SE (Jones et al., 2002; Pouliot et al., 2013; Zheng et al., 2010). The current studies were conducted to both at ASPET Journals on September 30, 2021 evaluate the anticonvulsant efficacy of IV -administered GNX with administration delays up to 1 h after seizure onset, and to establish a pharmacokinetic (PK)/ pharmacodynamics (PD) relationship that would differentiate GNX from existing treatments