Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Veterinary Clinical Sciences
Non Technical Summary
Expected results and relevance of dataAs stated in the hypotheses, we anticipated that curcumin and agmatine will improve limbuse similarly to phenylbutazone and more than placebo. Further, we expect all treatments toreach maximum levels within 2 hours of administration and each to have a half-life of about24 hours. Agmatine and curcumin are not expected to affect gastric ulceration whilephenylbutazone may exacerbate or initiate ulceration. Regardless of whether thesehypotheses are true, the results will be significant and relevant since the following will beestablished: 1) Appropriate dose, administration route and frequency of natural antiinflammatoryagents for horses; 2) Efficacy of two natural anti-inflammatory agents forequine forelimb OA pain compared to phenylbutazone; 3) Effects of two natural antiinflammatoryagents on gastric ulceration compared to phenylbutazone. The results of thisstudy will validate the methods and substantiate the hypotheses to extramural fundingagencies. They will be of significant interest and value to veterinarians and horse owners asby significantly augmenting current treatment options for horses affected by OA.Pitfalls and limitationsVariable response to therapy may limit the ability to detect differences among treatments.This will be largely mitigated by the cross over study design and distinct mechanisms ofaction among treatments. The dose and administration schedule may not be sufficient toreach meaningful therapeutic levels of curcumin or agmatine. Those selected for the studyare based on the manufacturer's recommendations to reduce the likelihood of this occurring.Enrollment of horses may take longer than anticipated. It is not necessary to enroll all horsesat the same time, and in fact, it will be more conducive to a reasonable workload if horses areenrolled incrementally. The methods and outcomes are routine in the laboratory and thereforeunlikely to interfere with completion of the objectives.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Osteoarthritis (OA) is a leading cause of lameness in equine patients. To date, no treatmentprevents progressive joint degeneration and treatment regimens often rely heavily onsystemic nonsteroidal anti-inflammatory drugs (NSAIDs) for pain management. However,there are complications associated with long-term administration of conventional NSAIDSincluding gastrointestinal (GI) ulceration and renal toxicity. Naturally derived antiinflammatoryand analgesic compounds like curcumin and agmatine may be safe alternativesto manage inflammation and pain associated with equine OA. The overall goals of theproposed investigations are to establish the serum half-life as well as the safety and efficacyof agmatine and curcumin in horses with naturally occurring forelimb OA compared to thegold standard NSAID phenylbutazone.
Project Methods
Twelve thoroughbred and/or quarter horse mares or geldings, 3-10 years, 400-500 kg, American Association of Equine Practitioners [AAEP] grade 2 to 4 lameness, and radiographic evidence of OA (fetlock, pastern, or carpus) in a single forelimb will be selected from the LSU Equine Health Studies Program Research and Teaching Herd. Using a randomized block design, horses will be assigned to receive 1 of 3 commercially available treatments or placebo in random order: 1) curcumin (1.06 g/horse, PO, sid, Longvida®); 2) agmatine (25 mg/kg, PO, sid, G-Agmatine brand®); 3) phenylbutazone (6.6 mg/kg, PO, sid, Equizone 100™); or placebo (4l grain, PO, sid). Each treatment be administered for 30 days with a 14-day period between (Fig. 1). Outcome measures including subjective lameness evaluation and joint pain, physical exam, blood chemistry and complete blood count (CBC), gastric ulcer evaluation via gastroscopy, and GRF quantification with a force platform will be conducted before (day 0) and after 15, and 30 days of treatment. Pharmacokinetics of curcumin, agmatine, and phenylbutazone will be evaluated before and after 15, 30, 45, 60, 75, 90, 105, 120, 150, 180 minutes, 6, 12, 18, and 24 hours, and 15 and 30 days of administration. Evidence of radiographic OA will be graded using established rubrics by a board-certified radiologist before each treatment phase11,12.B. Techniques to be employedRadiographs: Horses will be sedated with xylazine (5 mg/kg, IV; AnaSed®, Lloyd, Shenandoah, IA) for standard, orthogonal radiographs before inclusion in the study. Radiographic changes in the pastern, fetlock, or carpus of the affected limb will be scored according to published parameters by a board-certified radiologist unaware of treatment 11,12. Subjective Lameness and Joint Pain: Evaluations will be conducted between 2 and 6 hourscompound administration. Each horse will be observed in hand at a walk and a trot in a straight line and lunging in both clockwise and counterclockwise directions. Lameness will be scored by a board-certified surgeon unaware of the treatment phase according to the AAEP scoring system13. Range of motion (flexion) and response to palpation of the affectedjoint will be performed just prior to the subjective lameness evaluation.Blood Analyses and Pharmacokinetics: An intravenous catheter will be placed in a jugular vein of each horse for blood collection before and after 15, 30, 45, 60, 75, 90, 105, 120, 150, 180 minutes, 6, 12, 18, and 24 hours of each treatment. Blood will be collected byvenipuncture after 15 and 30 days of treatment. Blood samples will be centrifuged at 2500 g for 10 minutes at 4°C. Plasma will be stored in polystyrene tubes at -20°C until processing. The concentrations of curcumin, agmatine, and phenylbutazone will be measured by HPLCafter ether extraction, as previously reported (Eurofins Panlabs, Inc., St. Charles, MO)14. A CBC and chemistry panel will be performed on blood before and after 15 and 30 days of each treatment phase.Force Plate Analysis: Kinetic gait analysis will be performed on each horse using a 900 x 900 mm force platform embedded in the center of a 40 m concrete runway (Model #BP900900, Advanced Mechanical Technology, Inc., Watertown, MA). The force platform surface is the same color and texture as the runway. An experienced handler will trot horses for all trials. A trial is considered successful if a forelimb contacts the force platformfollowed by contact of the ipsilateral hind limb at a velocity of 2.00-3.80 m/s and acceleration of 0.9 to -0.9 m/s. A series of five retroflective photocell sensors (Mek92-PAD, Doslyn Clark Controls, Inc., Elizabethtown, NC) will be used to determine velocity andacceleration in each trial. The middle sensor is positioned so that it bisects the center of the force platform. Data logging is triggered by a force of 50 N. Trials are rejected if the hoof is not straight on the force platform, is not completely on the force platform or is within 5 cm ofthe force platform edge. The velocity and acceleration ranges selected include a comfortable trot for all of the subjects included in the study. There is little difference in velocity between sound and lame horses, and horses select a preferred stride length and frequency to obtain anenergetically optimal velocity. Based on this information, a speed is not imposed on the horses, but individual limb stance times are recorded. Five successful trials are recorded for each limb. Ground reaction forces are recorded at a rate of 100 Hz and processed withcommercially available software (Acquire V7.3, Sharon Software Systems, Bengaluru, India). Recorded forces include vertical peak force and impulse, both normalized to body weight. Mean vertical loading and unloading rates as well as stance time will be evaluated according to described methods. Mean values from each horse are used for statistical analyses of all kinetic outcome measures. Percent change in each GRF measure will be calculated as: Percent Change = [(GRF after treatment - GRF at baseline)/(GRF at baseline)]x 100.Gastroscopy: The stomach and proximal duodenum of each horse will be evaluated using a 3m endoscope (Karl Storz, El Segundo, CA) on days 0 and 30. Feed will be withheld for 12-24hours, but water will not be withheld prior to gastroscopy to improve visualization of thestomach. Horses will be sedated with xylazine (0.4 mg/kg, IV; AnaSed®, Lloyd, Shenandoah,IA). To enable observation of the non-glandular squamous mucosa (fundus ventriculi), margoplicatus, and glandular mucosa (corpus ventriculi), the stomach will be insufflated with airusing an air compressor (Airhead® High Pressure Air Pump, Kwik Tek, Denver, CO) attachedto the endoscope biopsy channel until the rugae, or stomach folds, disappeared. The mucosawill be rinsed of adherent food material and mucus with tap water flushed through the endoscope biopsy channel. Each horse's stomach will be assigned an equine gastric ulcersyndrome (EGUS) score according to established parameters by a board-certified internist unaware of treatment phase15. In addition, glandular ulcers will be counted and recorded.Analysis of resultsPower Analysis: A minimum sample size requirement calculation (one-way ANOVA) using80% power (β=0.20), a significance level of 0.05 (α=0.05), an effect size of 0.4 and a standarddeviation of 5% change in vertical peak force indicated 12 subjects per group.Statistical Analysis: Measures (GRFs, subjective lameness, EGUS score) will be comparedamong treatments with MANOVA models using horse, time, treatment, and OA score asfixed effects. All analyses will be conducted using commercially available software (SASStatistical Software). Repeated measures ANOVA models will be used to evaluatepharmacokinetic outcomes.