Larval Zebrafish Model for FDA-Approved Drug Repositioning for Tobacco Dependence Treatment

<div><p>Cigarette smoking remains the most preventable cause of death and excess health care costs in the United States, and is a leading cause of death among alcoholics. Long-term tobacco abstinence rates are low, and pharmacotherapeutic options are limited. Repositioning medications approved by the U.S. Food and Drug Administration (FDA) may efficiently provide clinicians with new treatment options. We developed a drug-repositioning paradigm using larval zebrafish locomotion and established predictive clinical validity using FDA-approved smoking cessation therapeutics. We evaluated 39 physician-vetted medications for nicotine-induced locomotor activation blockade. We further evaluated candidate medications for altered ethanol response, as well as in combination with varenicline for nicotine-response attenuation. Six medications specifically inhibited the nicotine response. Among this set, apomorphine and topiramate blocked both nicotine and ethanol responses. Both positively interact with varenicline in the Bliss Independence test, indicating potential synergistic interactions suggesting these are candidates for translation into Phase II clinical trials for smoking cessation.</p></div

Tobacco dependence treatment medications (varenicline, bupropion) alter the larval zebrafish nicotine response.

<p>(<b>A–C</b>) Larvae pretreated in varenicline (50 μM) overnight and challenged with stimulus at 6 dpf (± SE). Varenicline attenuates (<b>A</b>) 20 μM nicotine response, but not (<b>B</b>) 50 μM cinnamon oil or (<b>C</b>) 25 μM mustard oil response. (<b>D</b>) Acute treatment with 50 μM varenicline does not affect locomotion at 5 dpf (± SE). (<b>E–F</b>) Mean cumulative distance traveled in the 4 minutes post stimulus exposure as a percent of the average untreated stimulus response (± SE). Wash Nic = 24-hour washout period following acute nicotine experiment and re-tested at 7 dpf. Acute early and acute late response represents the first 4 minutes and last 4 minutes, respectively, post drug exposure at 5 dpf. (<b>E</b>) Movement quantitation for varenicline experiments. (<b>F</b>) Movement quantitation for bupropion experiments. n≥30 larvae per condition; *  = p<0.05; Students t-test.</p

Combination therapy assessment with varenicline.

<p>One known and two new potential synergistic interactions with varenicline have been identified. Bupropion and topiramate show a positive interaction with varenicline using Bliss Independence analysis. Larval zebrafish were pretreated in each medication and varenicline at 50% of the monotherapy dose found to be effective. Six replicates of 10 larvae per condition were challenged with 20 μM nicotine. Conditions included drug pretreated, varenicline pretreated, drug and varenicline pretreated and untreated larvae. We calculated the expected effect of each combination with the equation: E = D×V, where D =  % nicotine response of drug treated larvae and V =  % nicotine response of varenicline treated larvae. The experimental percent nicotine response is plotted against the theoretical calculated response. Apomorphine, bupropion, and topiramate show a greater-than-additive effect, diazepam and betaxolol have an additive effect, and carisoprodol, clonazepam, lorazepam, and zolpidem have a less-than-additive effect on locomotor response to nicotine.</p

Nicotine-modulating FDA-approved drugs show distinct effects on ethanol-induced locomotor activation.

<p>(<b>A</b>) Inverted-U dose response on locomotion from 0 to 4% ethanol. Cumulative distance is the mean of the summed one-second distances 30–40 minutes post bath ethanol exposure (± SE); n = 30 larvae per condition. (<b>B</b>) Ethanol (1.5%)-induced locomotion is decreased with overnight pretreatment in disulfiram (500 nM); n = 60 larvae per condition (± SE). (<b>C</b>) Larvae pretreated overnight in medication are subsequently challenged with 1.5% ethanol. Bars represent mean cumulative distance traveled during the 30–40 minutes post-ethanol exposure as a percent of the average untreated ethanol response (± SE); n = 60 larvae per condition; *  = p<0.05; † = 0.05</p

Evaluating FDA-approved medications identifies new modifiers of the nicotine response using larval zebrafish locomotion.

<p>Zebrafish pretreated with medication overnight and challenged with 20 μM nicotine. Category (i) compounds: no attenuation of the nicotine response and no toxicity with overnight incubation in the drug up to 1 mM concentrations. Category (ii) compounds: no attenuation of the nicotine response, but toxicity was observed at the next highest dose evaluated. Category (iii) compounds: significantly attenuated the nicotine response and the cinnamon oil and/or mustard oil response. Category (iv) compounds: significantly attenuated the locomotor response to nicotine, but not to cinnamon oil or mustard oil responses. Current Treatment  =  FDA-approved medications for smoking cessation. n≥30 larvae per condition; *  = p<0.05; † = 0.05</p

Distinct behavioral responses by FDA-approved medications on the nicotine and ethanol locomotor response.

<p>The ability to modulate the ethanol (Y-axis) or nicotine (X-axis) locomotor responses is shown. Apomorphine and topiramate attenuate both nicotine and ethanol responses.</p

Larval zebrafish respond to nicotine. Increased locomotion occurs immediately following full-body exposure in 20 μM nicotine.

<p>(<b>A</b>) The inverted-U dose response. (<b>B–C</b>) Pretreatment with nAChR antagonists with nicotine challenge (± SE): (<b>B</b>) mecamylamine (10 μM), (<b>C</b>) hexamethonium (5,000 μM, p = 0.803), a peripheral nervous system-only nAChR antagonist. (<b>D</b>) Mean cumulative distance traveled in the first 4 minutes post-nicotine exposure as a percent of the average untreated nicotine response (± SE). n≥30 larvae per condition; *  = p<0.05; Students t-test.</p

Discrete event simulation for performance modelling in healthcare: a review of the literature

Discrete Event Simulation (DES) has been widely used in modelling health-care systems for many years and a simple citation analysis shows that the number of papers published has increased markedly since 2004. Over the last 30 years several significant reviews of DES papers have been published and we build on these to focus on the most recent era, with an interest in performance modelling within hospitals. As there are few papers that propose or illustrate general approaches, we classify papers according to the areas of application evident in the literature, discussing the apparent lack of genericity. There is considerable diversity in the objectives of reported studies and in the consequent level of detail: We discuss why specificity dominates and why more generic approaches are rare