Characterization of a Novel Repeat-Dosing Model for Preclinical Diazepam Studies
Abstract number :
2.245
Submission category :
7. Anti-seizure Medications / 7A. Animal Studies
Year :
2023
Submission ID :
597
Source :
www.aesnet.org
Presentation date :
12/3/2023 12:00:00 AM
Published date :
Authors :
First Author: Sunita Misra, MD, PhD – Neurelis, Inc., San Diego, CA
Presenting Author: Michelle Guignet, PhD – School of Pharmacy, University of Washington, Seattle, WA, USA
Michelle Guignet, PhD – Department of Pharmacy, University of Washington, Seattle, WA; H. Steve White, PhD – Department of Pharmacy, University of Washington, Seattle, WA; Enrique Carrazana, MD – Neurelis, Inc., San Diego, CA; University of Hawaii, John A. Burns School of Medicine, Honolulu, HI; Adrian Rabinowicz, MD – Neurelis, Inc., San Diego, CA
Rationale: Diazepam (DZP) has been used for decades as an antiseizure medicine, and its safety, efficacy, and pharmacokinetic profiles are well described. However, DZP elimination pharmacokinetics is substantially faster in rats than humans, complicating subchronic and chronic preclinical studies in rats. Few preclinical studies have examined drug distribution and prolonged clearance with repeat dosing of DZP. Previous rat studies suggest that repeat doses of intraperitoneal (IP) DZP 20 mg/kg result in sustained DZP accumulation in serum and cerebrospinal fluid, but the utility of the findings was limited because the concentration was associated with impaired neurological function (ie, loss of righting reflex). The purpose of this study was to identify a suitable DZP dosing strategy in rats for subsequent subchronic in vivo efficacy studies.
Methods: Adult male Sprague Dawley rats (~400 g) were administered three repeat doses of IP DZP (0.75, 1.5, 3, or 6 mg/kg) one hour apart. A separate group received only a single 3-mg/kg dose. Righting reflex was evaluated before administration of each dose. Blood was collected at 10 minutes and one, three, or six hours after the final injection. Brain samples were collected at one, three, or six hours after the final DZP injection. DZP levels from plasma and brain were determined using liquid chromatography–mass spectrometry. Differences in plasma and brain DZP concentrations at different doses were tested for significance by 2-way analysis of variance with main effects only.
Results: Repeat dosing of DZP (0.75–3 mg/kg) resulted in a dose-dependent increase in plasma and brain DZP levels up to 6 hours postdose (Figure 1A–B; n=3–6). This dosing protocol was associated with a normal righting reflex; however, a single 6-mg/kg dose resulted in substantial impairment and was not studied further. For the 3-mg/kg dose, plasma DZP levels (mean±SD) at 1 and 3 hours were 180±156 ng/mL and 176±56 ng/mL, respectively, whereas brain DZP levels were 140±126 ng/g and 161±62 ng/g. Brain-to-plasma ratios suggest a modest accumulation of DZP in the brain as long as 6 hours after the last dose (Figure 1C). In addition, brain accumulation of DZP at 6 hours was greater in rats that received repeat doses compared with a single dose (38.2±34.6 ng/g vs 6.1±3.4 ng/g).
Conclusions: In this rat model, a repeat-dosing paradigm of DZP at doses up to 3 mg/kg resulted in sustained and measurable DZP levels in plasma and brain, which were similar to plasma concentrations observed in humans. Accumulation of DZP was observed in rat brain tissue compared with plasma, suggesting that DZP may be long-acting at the site of action. This repeat-dosing paradigm for DZP in rats mimics drug concentrations and accumulation seen in humans, offering a preclinical tool to study the impact of benzodiazepine rescue therapy on seizure cluster biology.
Funding: Neurelis, Inc.
Anti-seizure Medications