Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Veterinary Medicine & Surgery
Non Technical Summary
Laminitis ('founder') is a painful and disfiguring disease of horses that frequently necessitates lowering of athletic performance targets or, in severe cases, euthanasia. In spite of significant research, there still does not exist an effective therapeutic agent for the purpose of preventing laminitis in at risk horses (such as those affected with gastrointestinal disturbances). The main purpose of this project is to develop and validate a method by which horses can be treated with lithium chloride in a manner that predictably delivers a therapeutic plasma concentration of lithium in the known therapeutic range. Such a method will involve the injection of an IV bolus of lithium chloride, followed by a continuous rate infusion of lithium chloride using a 'pump'. Our challenge is to determine the exact dose of lithium chloride to be used and the rate at which it should be delivered. This task can be accomplished by measuring plasma lithium concentrations in healthy horses following a single IV bolus of the drug - thus developing a pharmacokinetic (PK) profile. With the help of a computer, using these results, we can calculate the optimal and logical doing schedule that will work to assure therapeutic lithium concentrations but also avoid toxic (too high) levels. Therefore, we plan to try to dose some horses with the PK-derived dosing schedule and to ascertain whether it is effective (or not). It will be important to show that, with success, we are able to maintain the appropriate plasma lithium concentration in the therapeutic range for 30 hours because this is the time frame in which horses being treated for colic or diarrhea are at risk for developing laminitis. We will also validate the commonly used plasma lithium assay (as broadly used in the human medical field) for horses. This is important because it will be necessary to monitor the lithium concentration in horses during treatment. If our approach is successful, we would plan to seek funds to employ lithium therapy for the purpose of preventing laminitis in an experimental model of the disease. Lithium therapy has not hitherto been used for treatment of horses. We believe that this treatment may provide the first effective therapeutic method by which veterinarians can effectively prevent laminitis in at-risk horses.We have assembled a team of veterinary clinician scientists with specific areas of expertise in order to maximize the chance for a successful outcome. Drs. Philip Johnson and Dorothy Whelchel are equine veterinary internal medicine specialists with considerable experience working with horses in critical conditions (such as those with severe colic and diarrhea). Dr. Johnson has extensive experience working with laminitis, specifically. Dr. Alex Bukoski is a veterinary anesthesiologist with excellent training in pharmacology and the generation/interpretation of pharmacokinetic data. Drs. Chuck Wiedmeyer and Tim Evans are specialists in the fields of clinical pathology and toxicology, respectively. Dr. Wiedmeyer has already developed the commonly used (humanoriented) plasma lithium assay and it seems to be working well. Dr. Evans will facilitate comparisons between Dr. Wiedmeyer's results and those of a 'gold standard' method for determination of lithium concentration in blood, thus validating the former.
Animal Health Component
35%
Research Effort Categories
Basic
15%
Applied
35%
Developmental
50%
Goals / Objectives
These studies are essential steps for the purpose of eventually validating LiCl as an effective prophylactic for laminitis.Specific objectivesObjective 1: Obtain pharmacokinetic data following a single IV injection of lithium chloride by measuring the plasma lithium concentration following administration of a bolus of lithium chloride solution to healthy adult horses.Objective 2: Validate that the routine clinical laboratory spectrophotometric enzymatic assay used to measure plasma lithium concentration (for other species, such as humans) is accurate for the purpose of determining plasma lithium concentrations in equine blood.Objective 3: Demonstrate that, based on PK data from Objective 2, using variable (calculated) doses of lithium chloride administered via CRI over the course of 30 h, the plasma lithium concentration will be maintained within the therapeutic range.
Project Methods
Objective 1: Obtain PK data for disposition of lithium following administration of an IV bolus. Horses - Eight healthy adult horses that belong to the UMC equine teaching herd will be selected. Each horse will be transported from Middlebush Farm and accommodated in stalls in the VMTH for the purpose of the study. All horses will be weighed and fed a standardized ration of grass hay and ad libitum access to water before and during each investigative period. Horses will be acclimated in the VMTH for at least 3 days prior to onset of the study. On the day prior to administration of the lithium chloride bolus, each horse will be equipped with an IV catheter placed aseptically in both the left and right jugular veins using lidocaine for local anesthesia (tranquilizer drugs will not be used). Catheters will be managed routinely using a hygienic approach and with regular flushing with heparinized saline (q 6 h).Lithium chloride bolus - Following collection of baseline blood samples, lithium chloride (0.15 mmol/kg of body weight; 75 mmol per 500 kg horse) will be injected as a bolus over the course of 2 minutes via the catheter in the right jugular vein. Sampling and sampling times - At each sampling time, blood collected from the catheter in the left jugular vein will be injected into green-top (heparinized) 20-ml Vacutainer™ tubes. At each sampling, 15 ml of blood will be drawn and discarded (to assure that residual blood in the catheter is not being sampled). Plasma will be derived by centrifugation and stored at -80°C until analysis as a batch. Blood samples will be collected at the following times with respect to the injection of lithium chloride: minus-30 min, 0 time, +2.5 min, +5 min, +10 min, +20 min, +40 min, +60 min, +2 hr, +4 hr, +8 hr, +12 hr, +24 hr, and +48 hr.Horse observations - Each horse will be critically evaluated at each blood sampling time point (direct observation and by moving the horse around in the stall) for evidence of any neurological signs (weakness, ataxia, abnormal behavior) that might occur consequent to lithium chloride treatment. Allhorses will receive full physical examinations (included rectal temperature, heart and respiratory ratesevery 6 h).Objective 2: Validate that the routine clinical laboratory spectrophotometric enzymatic assay used to measure plasma lithium concentration (for other species, such as humans) is accurate for the purpose of determining plasma lithium concentrations in equine blood. The concentration of lithium in plasma from each sample time will be measured using a routine clinical laboratory method (spectrophotometric enzymatic assay) and by inductively coupled plasma mass spectrometry (gold standard).The lithium-specific spectrophotometric enzymatic assay has been developed using equipment in Dr. Wiedmeyer's laboratory and details of its approach are available here [Diazyme Liquid Stable Lithium Enzymatic Assay™]. Briefly, lithium is determined spectrophotometrically through a kinetic coupled enzyme assay system involving a Diazyme™ proprietary phosphatase whose activity is sensitive to lithium (IC50=0.1 mM). Through enzymatic coupling, the phosphatase substrate is converted to hypoxanthine by a series of enzymatic reactions to generate uric acid and hydrogen peroxide (H2O2). H2O2 generated reacts with N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (EHSPT) and 4-aminoantipyrine (4-AA) in the presence of peroxidase (POD) to form a quinone dye that has maximal absorbance at 556 nm. The rate of the quinine dye formation is inversely proportional to the concentration of lithium in serum samples.Inductively coupled plasma mass spectrometry (ICP-MS) is a type of mass spectrometry that is capable of routinely detecting metals (and several non-metals) at concentrations as low as one part in Lithium for equine laminitis prevention 109 (part per billion, ppb) on non-interfered low-background isotopes. This method is achieved by digesting the serum sample in acid, ionizing the sample with inductively coupled plasma, and then using a mass spectrometer to separate and quantify those ions. Appropriate spikes and duplicates will be run for QA/QC purposes.Data analysis - The concordance coefficient will be calculated to address the question of agreement between the 2 measurement methods.40 The concordance coefficient is more appropriate than a simple correlation coefficient because the latter is a measure of the strength of the linear relationship between the 2 measures but does not address whether they actually agree (ie, yield the same response). The mixed procedure in the statistical software SAS v9e will be used because it allowed fitting a model with repeated observations on the same subject as well as heterogeneous variances. The null hypothesis will be refuted if p<0.05.Although we (UMC-CVM) do not have direct access to ICP-MS for purposes of determining lithium concentration in plasma samples, Dr. Evans has identified a laboratory at Utah State University that will be able to undertake this analysis. Therefore, plasma samples will be shipped to the Utah State University laboratory for measurement of lithium concentration by ICP-MS. Results from each method will be compared in a "comparison plot" that displays one test result on the y-axis versus the comparison result on the x-axis. As points are accumulated, a visual line of best fit will be drawn to show the general relationship between the 2 methods and help identify discrepant results. Identificationof any discrepant results evident from the graph will require re-analysis of affected samples.Objective 3: Demonstrate that, based on PK data from Objective 2, using variable (calculated) doses of lithium chloride administered via CRI over the course of 30 h, the plasma lithium concentration will be maintained within the therapeutic range. Following completion of Objectives 1 and 2, we will have data upon which a dosing plan can be reasonably based.Dr. Bukoski has already worked on lithium dosing protocols in an attempt to produce a staged treatment plan (administration of lithium chloride boluses alongside a variable rate of lithium chloride CRI) intended to maintain a plasma lithium concentration in the therapeutic range for 30 h. However, it became obvious that this pharmacological planning required PK data (hitherto unavailable) obtained following an IV LiCl bolus (as in Objective 1) (our earlier efforts had been frustrated by the fact that the only other published study was based on PK data obtained following an IV LiCl infusion administered over a long period of time). Using the bona fide ('bolused') Li PK data generated from Objective 1, Dr. Bukoski will develop a treatment protocol based on the weight of the targeted horse. For this purpose, we will require special software (Matlab optimization toolkit which includes functions for nonlinear optimization). We envision that the treatment protocol will entail starting with an IV bolus of lithium chloride followed up with a CRI with decreasing rates of infusion. An example of a lithium dose scheme that we developed (for a 24-hour period) is available for inspection in the 'preliminary results' section. We propose to use the derived PK results to develop a dosing schedule and then undertake a pilot study (6 horses) in which each horse will be treated with the planned lithium chloride dosing schedule and the outcome will be assessed based on frequent plasma lithium determinations during the treatment.