A Pharmacological Screening Approach for Discovery of Neuroprotective Compounds in Ischemic Stroke

<div><p>With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-β-hydroxysteroid dehydrogenase-1 (11β-HSD1), gave rise to similarly potent <i>in vivo</i> neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11β-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.</p></div

Treatment with Carbenoxolone attenuates ischemic brain injury.

<p>Animals were subjected to 90 minutes of tMCAo and were treated 5 minutes pre-tMCAo and 3 hours post-tMCAo with Carbenoxolone or vehicle (H₂O) at the indicated total doses. Total infarction size was significantly decreased in tMCAo animals treated with 40 mg/kg (n = 5) (**p<0.01) and 60 mg/kg (n = 8) (**p<0.01) as compared to the 10 mg/kg treated group (n = 7) as well as in the 60 mg/kg (n = 8) (*p<0.05) group as compared to the vehicle group (n = 19). Data were assessed using the Kruskal-Wallis test and significant results from Dunn’s Multiple Comparison test are shown. Box plots represent median and quartiles and whiskers show minimum and maximum values.</p

Carbenoxolone attenuates delayed OGD-induced hippocampal cell death.

<p>Hippocampal slice cultures were exposed to oxygen-glucose deprivation (OGD) and stained with Propidium iodide (PI). Photographs of the control and OGD slices at pre- and 24 hours post-OGD. A representative image is shown for each experiment (n = 4). MK-801 (Dizocilpine) was used as a positive control (<b>a</b>). The compounds were added 2 hours prior to the OGD insult. Mean fluorescence intensity (MFI) was measured 24 hours post-OGD. Both 10 µM Carbenoxolone (n = 12) (**p<0.01) and 10 µM MK-801 (n = 12) (***p<0.001) significantly reduced cell death compared to the vehicle group (n = 12) (<b>b</b>). Data were assessed using the Kruskal-Wallis test and significant results from Dunn’s Multiple Comparison test are displayed. Box plots represent median and quartiles and whiskers show minimum and maximum values.</p

Molecular structure and neuroprotection of Carbenoxolone.

<p>Molecular structure of Carbenoxolone, a synthetic derivative (succinyl ester) of Glycyrrhetinic acid (constituent of licorice). Carbenoxolone is an inhibitor of 11β steroid dehydrogenase enzymes (HSD1 and HSD2) and gap junctions (<b>a</b>). Protection against OGD-induced neuronal damage by Carbenoxolone. Primary cortical neurons were subjected to 2 hours of OGD and neuronal damage was assayed using the Cell Titer Glo assay at 24 hours of recovery, in presence of vehicle, 10 µM Carbenoxolone pre-during-post (PDP) (***p<0.001 vs. Vehicle; n = 10–13), or exclusively post OGD (Post) (*p<0.05 vs. Vehicle; n = 10–13). Carbenoxolone demonstrated neuroprotective activity in both PDP and post treatment experiments (n = 10–13) (<b>b</b>). Data were assessed via one-way ANOVA and significant results of the Dunnett’s post-test are shown with lines representing mean.</p

Neuroprotection and class of screened compounds.

<p>A library of pharmacologically active compounds was screened using an oxygen-glucose deprivation (OGD) assay with primary cortical neurons to identify neuroprotective compounds (<b>a</b>). At 24 hours post-OGD, approximately 50% of the 880 screened compounds showed neuroprotection at levels over 50% compared to controls (<b>a</b>). Compounds that showed protection represent an array of pharmacological classes including antibacterial, anti-inflammatory, anti-coagulant, and anti-hyperlipidemic compounds (<b>b</b>). The complete list of compounds tested and the degree of protection is displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069233#pone.0069233.s001" target="_blank">Table S1</a>.</p

Molecular structure and neuroprotection of BVT-2733.

<p>Molecular structure of the specific 11β-HSD1 inhibitor, BVT-2733 (3-chloro-2-methyl-N-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethyl) thiazol-2-yl) benzenesulfonamide hydrochloride) (<b>a</b>). Animals were subjected to 90 minutes of tMCAo and were treated with BVT-2733 30 mg/kg or vehicle (PEG 500 20%, DMSO 4%) at 3 hours and 7 hours post-reperfusion, for a total dosage of 60 mg/kg. Treatment with BVT-2733 (IP, intraperitoneal) (n = 10–11 in each treatment group) attenuated the ischemic brain injury (<b>b</b>). Data were assessed using an unpaired Student’s t-test. Scatter plots with mean values and significance is shown. Representative images of brain sections of treated animals: Neuronal damage was quantified by TTC staining; white indicates infarction and red staining indicates normal tissue (<b>c</b>).</p

List of compounds displaying post-injury neuroprotective activity in the oxygen glucose deprivation (OGD) assay in cortical neurons.

<p>List of compounds displaying post-injury neuroprotective activity in the oxygen glucose deprivation (OGD) assay in cortical neurons.</p

List of compounds displaying post-injury neuroprotective activity in the NMDA-induced toxicity assay in cortical neurons.

<p>List of compounds displaying post-injury neuroprotective activity in the NMDA-induced toxicity assay in cortical neurons.</p

Profiling flow-chart to identify neuroprotective compounds with potential therapeutic efficacy.

<p>Profiling flow-chart to identify neuroprotective compounds with potential therapeutic efficacy.</p

Post treatment in vivo efficacy of Carbenoxolone.

<p>Animals were subjected to 90 minutes of tMCAo treatment with 60 mg/kg total dose at 3 hours (30 mg/kg) and 6 hours (30 mg/kg) post-MCAo. Neuronal damage was quantified by TTC staining (n = 8–45), white (infarction), red (normal tissue) (<b>a</b>). Exploration of Carbenoxolone therapeutic window post-MCAo injury: Carbenonxolone was administered at a 60 mg/kg total dose (2×30 mg/kg) with a 3 hour interval with the first dose delivered at 1.5, 3, or 6 hours post-treatment (tx = treatment). The injuries in all the groups were quantified by TTC staining at 24 hours post injury (<b>b</b>). Data were assessed via one-way ANOVA and significant results of the Dunnett’s post-test and means are shown.</p