Recipient Organization
UNIV OF CALIFORNIA (VET-MED)
(N/A)
DAVIS,CA 95616
Performing Department
VM: Surg/Rad Science
Non Technical Summary
Conventional drugs used for pain relief, sedation, anesthesia, or euthanasia in livestock can leave harmful residues in food or the environment. L-carvone and methyl salicylate are edible compounds found in mint plants that are generally recognized as safe. Yet, preliminary work in our lab has shown these mint extracts can affect cellular targets known to be important for anesthetic and analgesia activity. Moreover, our pilot studies in laboratory rodents demonstrate that these mint extracts can produce general anesthesia or euthanasia. In the project, we predict that L-carvone and methyl salicylate can produce analgesia, anesthesia, and euthanasia in sheep, and that chemically-related compoundscan show similar or even greater effiects atcell receptors responsible for these effects. In Aim 1, we will administer L-carvone or methyl salicylate via different routes to adult sheep, measure drug concentrations in blood over time, and fit data to a pharmacokinetic model describing drug uptake, distribution, and elimination. In Aim 2, we will study the pharmacologic and physiologic effects of L-carvone and methyl salicylate in sheep. First, we will measurethe blood concentration of each drug required to prevent movement in response to noxiousstimuli. Analgesic efficacy will be tested using devices designed to elicit behavioral responses to temperature or pressure stimuli, and these behavioral responses will becorrelated to blood drug concentrations. Euthanasia will be studied by measuring electroencephalographic and cardiorespiratory responses following IV bolus injection of each compound. In Aim 3, we will use standard cell electrophysiology methods to study the effects of other plant-derived relativesof L-carvone and methyl salicylateon ion channels important to analgesic and anesthetic activity. This will reveal drug structure-activity relationships key to identifying new pharmaceutics of food origin.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
In pilot studies, we have previously shown that certain mint compounds affect cellular targets improtant in mediatingpain relief or anesthesia, and we have demonstrated that administration of these compounds to rodents can be used to produce general anesthesia (or at higher doses, euthanasia).The goal of this project is to determine whether these same mint extractsare efficacious as analgesics, general anesthetics, and/or euthanasia agents in a food-producing animal model (sheep). This project will also investigate whether other chemically-related plant extracts exhibit similar or greater efficacy at cellular targets important for pain relief or anesthesia.
Project Methods
Aim 1: Pharmacokinetics (PK) Aim 1a will measure L-carvone pharmacokinetics in 6 sheep, and Aim 1b will measure methyl salicylate pharmacokinetics in 6 sheep. For IV studies, both jugular veins will be catheterized, and baseline (pre-drug) blood collected for complete blood count (CBC) and serum chemistry. Next, L-carvone or methyl salicylate in Intralipid will be injected as an IV bolus (time= 0min) at the dose determined in the dose-finding study described previously. Blood will be sampled from the contralateral jugular vein at 1, 2, 4, 8, 16, 32 min and 1, 2, 4, and 8 hours after administration in heparin tubes, and plasma will be separated by centrifugation. During this time, body position (standing, recumbent), consciousness (response to non-painful arousal, if recumbent), and anesthesia (response to coronary clamp, if unconscious) will be tested at each blood withdrawal point in order to correlate behavioral endpoints with plasma drug concentrations. Sedation will also be scored using an ordinal scale.Additional blood will be collected at 16 min and 8 hours after drug administration for CBC and chemistry analysis to look for evidence of drug-induced injury by comparison to baseline values. Sheep will be then euthanized by an AVMA-approved method, and muscle, liver, kidney, and fat tissues will be collected for measurement of drug L-carvone or methyl salicylate concentrations. Similar methods will be used to measure L-carvone and methyl salicylate pharmacokinetics (and associated behavioral effects) following intramuscular (IM) and subcutaneous (SC) injection.Aim 2: PharmacodynamicsAnesthetic Efficacy and Physiologic Responses (Aim 2a) In Aim 2a, anesthesia in 6 sheep will be induced and maintained with L-carvone or methyl salicylate prepared in 20% Intralipid for a total of 60 minutes, a duration suitable for common surgical or restraint procedures that may be performed under field conditions. Sheep will be fasted for 24 hours prior to general anesthesia. Both jugular veins will be catheterized: one for drug administration and the other for blood sampling. Blood will be collected for baseline CBC and chemistry measurement. Plasma concentrations (Cp) associated with general anesthesia in the Aim 1 pharmacokinetic study will be identified and targeted in this Aim 2a study. Once anesthesia is induced, intubatedsheep will be instrumented with an EKG, oscillometric non-invasive blood pressure monitor, pulse oximeter, capnometer, and temperature probe. Additionally, a 16-lead EEG plus 3-lead electrooculogram, and 2-lead electromyogram will be placed. A Nihon Kohden digital EEG system with video will be used to obtain all standard EEG recordings, a fast Fourier transform analysis (FFT) will be used to calculate absolute power (in μV2) in each frequency band (δ, θ, α, β, γ), and visual inspection of the raw EEG will be used to identify any electrical seizure activity. During anesthesia, physiologic responses will be recordedevery 5 minutes and blood collected for drug concentration measurement. At this time, a clamp will be applied to the coronary band to test for non-reflex movement. If movement is present/absent, the drug infusion rate will be increased/decreased by 15% and continued for 5 minutes. This process will be repeated throughout the 1-hour anesthetic duration. After 1-hour, blood will be collected for CBC and chemistry, the infusion will be stopped, and the sheep will be recovered from general anesthesia. Time to standing recovery and recovery quality will be recorded. Blood will be collected for drug analysis, CBC, and chemistry 8 hours after anesthetic induction. Body weight and feed intake will be monitored over the next 48 hours. At 2-days post-anesthesia, the sheep will be euthanized, and a necropsy will be performed to look for evidence of gross tissue injury. Muscle, fat, liver, and kidney samples will be collected for histopathologic examination and for measurement of tissue drug concentrations.Aim 2: PharmacodynamicsAnalgesic Efficacy for Thermal & Mechanical Pain (Aim 2b) In Aim 2b, L-carvone or methyl salicylate prepared in a propylene glycol solution will be administered intramuscularly 4 times in order to target plasma drug concentrations associated with an increase in electrical nociceptive threshold as described for the intramuscular pharmacokinetic methods of Aim 1. The IM drug pharmacokinetic model will be used to optimize initial dosing, and dosing interval will equal the time required for electrical nociceptive threshold to return to baseline levels. In 6 sheep studied per drug, a catheter will be placed in a jugular vein for blood sampling, and the hair over both metatarsi clipped. Amechanical nociceptive test device, consisting of a pin in a diaphragm actuator will be secured to one metatarsus. Athermal nociceptive testing device, consisting of a heating element and temperature sensor, will be secured to the contralateral metatarsus. The sheep will then be allowed to move freely in a stall enclosure between measurements, blood sampling, and drug dosing. Following baseline measurements, the first dose of drug will then be administered IM, and mechanical and thermal nociceptive thresholds will be quantified at 4 equally spaced time intervals for the expected analgesic duration previously determined during the Aim 1 IM pharmacokinetic study. At each analgesic test point, blood will be collected for drug analysis. The 2nd, 3rd, and 4th analgesic doses will be administered IM at the average time interval previously measured for Aim 1 electrical nociceptive thresholds to return to baseline responses. The nociceptive testing and blood sampling will be repeated for each subsequent dose. When nociceptive thresholds return to baseline after the 4th test, animals will be euthanized by an AVMA-approved method, and muscle, liver, kidney, fat, and injection site tissue will be harvested for measurement of drug concentrations using methods described in Aim 1.Aim 2: PharmacodynamicsEuthanasia Efficacy and Physiologic Responses (Aim 2c) In 6 sheep studied per drug, a jugular venous catheter will be placed, and the sheep will be instrumented with EEG scalp electrode and EKG as described previously for the anesthesia monitoring in Aim 2a. After baseline heart rate, respiratory rate, and EEG signals are collected, L-carvone or methyl salicylate prepared in Intralipid emulsion will be administered as an IV bolus at the dose previously described in the dose-finding study above. Time to recumbency, time to apnea, and time to cardiac arrest will be recorded, and any muscle twitching or vocalizations will be noted. Time to EKG asystole and time to EEG silence will be recorded. The EEG will also be analyzed by Fourier signal transformation for spectral power and by visual inspection of raw signals, as previously described in Aim 2a. Once the sheep is euthanized, we will harvest muscle, liver, kidney, and fat to measure drug concentrations using methods described in Aim 1.Aim 3: Receptor Efficacy of Other Terpenoids and Salicylate EstersIn vitro studies will use standard two-electrode voltage clamping techniques similar to those previously published by our laboratory. In these studies, intranuclear DNA injection or intracytoplasmic RNA injection of genes encoding channel subunits for the test channel (GABAAreceptor, NMDA receptor, or Nav1.2 channels) are made into Xenopus frog oocytes, a very large animal cell that does not express native anesthetic-sensitive ion channels. Two-electrode voltage clamp studies are conducted in a 0.25mL linear perfusion chamber. Dose-dependent electrophysiologic responses tokey related plant-derived terpenoid and salicylate esters will then be measured.