Andrew R. McKinstry-Wu

Discovery of a Novel General Anesthetic Chemotype Using High-throughput Screening

Background:The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Methods:Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene–apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for &ggr;-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. Results:From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. Conclusion:The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

Protocol for the Reconstructing Consciousness and Cognition (ReCCognition) Study.

Important scientific and clinical questions persist about general anesthesia despite the ubiquitous clinical use of anesthetic drugs in humans since their discovery. For example, it is not known how the brain reconstitutes consciousness and cognition after the profound functional perturbation of the anesthetized state, nor has a specific pattern of functional recovery been characterized. To date, there has been a lack of detailed investigation into rates of recovery and the potential orderly return of attention, sensorimotor function, memory, reasoning and logic, abstract thinking, and processing speed. Moreover, whether such neurobehavioral functions display an invariant sequence of return across individuals is similarly unknown. To address these questions, we designed a study of healthy volunteers undergoing general anesthesia with electroencephalography and serial testing of cognitive functions (NCT01911195). The aims of this study are to characterize the temporal patterns of neurobehavioral recovery over the first several hours following termination of a deep inhaled isoflurane general anesthetic and to identify common patterns of cognitive function recovery. Additionally, we will conduct spectral analysis and reconstruct functional networks from electroencephalographic data to identify any neural correlates (e.g., connectivity patterns, graph-theoretical variables) of cognitive recovery after the perturbation of general anesthesia. To accomplish these objectives, we will enroll a total of 60 consenting adults aged 20–40 across the three participating sites. Half of the study subjects will receive general anesthesia slowly titrated to loss of consciousness (LOC) with an intravenous infusion of propofol and thereafter be maintained for 3 h with 1.3 age adjusted minimum alveolar concentration of isoflurane, while the other half of subjects serves as awake controls to gauge effects of repeated neurobehavioral testing, spontaneous fatigue and endogenous rest-activity patterns.

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats. Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Development and validation of brain target controlled infusion of propofol in mice

Mechanisms through which anesthetics disrupt neuronal activity are incompletely understood. In order to study anesthetic mechanisms in the intact brain, tight control over anesthetic pharmacology in a genetically and neurophysiologically accessible animal model is essential. Here, we developed a pharmacokinetic model that quantitatively describes propofol distribution into and elimination out of the brain. To develop the model, we used jugular venous catheters to infuse propofol in mice and measured propofol concentration in serial timed brain and blood samples using high performance liquid chromatography (HPLC). We then used adaptive fitting procedures to find parameters of a three compartment pharmacokinetic model such that all measurements collected in the blood and in the brain across different infusion schemes are fit by a single model. The purpose of the model was to develop target controlled infusion (TCI) capable of maintaining constant brain propofol concentration at the desired level. We validated the model for two different targeted concentrations in independent cohorts of experiments not used for model fitting. The predictions made by the model were unbiased, and the measured brain concentration was indistinguishable from the targeted concentration. We also verified that at the targeted concentration, state of anesthesia evidenced by slowing of the electroencephalogram and behavioral unresponsiveness was attained. Thus, we developed a useful tool for performing experiments necessitating use of anesthetics and for the investigation of mechanisms of action of propofol in mice.

Xenon Anesthesia and CT: Noninvasive Measures of Brain Anesthetic Concentration

The existence of a barrier between anesthetic behavioral state transitions has been observed across phyla, but demonstrating that such a barrier exists and is not a pharmacokinetic artifact has not yet been possible in humans. Such an investigation requires temporally precise information regarding the brain concentration of anesthetic in order to demonstrate the specific pharmacokinetic-pharmacodynamic mismatch that is hysteresis. We propose a method to noninvasively determine brain tissue anesthetic concentration using computerized tomography and the radiopaque gaseous anesthetic xenon. Such a technique can be used to investigate pharmacokinetic-pharmacodynamic mismatches in humans.

Optoanesthesia: Use of Anesthetic Photolabels In Vivo

Investigation of how anesthetics produce hypnosis requires knowledge of their effects at the molecular, neuronal, circuit, and whole-brain network level. Anesthetic photolabels have long been used to explore how anesthetics bind and affect known protein targets, but they could potentially assist in investigation of anesthetic effects at higher organizational levels of the central nervous system. Here, we advocate the use and provide detailed methods for the application of anesthetic photolabels in slice electrophysiology and in intact animals as a means of investigating anesthetic effects on distinct circuits and brain centers.