Sixteen exercised healthy 4–7-year-old University owned Thoroughbred horses (8 mares and 8 geldings) were studied. The horses utilized for this study were part of an exercised research herd and were regularly exercised five days week utilizing a combination of workouts on a high speed-treadmill and Equineciser, following standard protocols established by our laboratory and described previously . Due to repeated arthrocentesis, LPS administration and induced lameness, horses were not exercised during the study period.
Horses did not receive any medications for a minimum of four weeks prior to commencement of the study and were determined healthy by physical examination, complete blood count (CBC) and a serum biochemistry panel performed the day before the first LPS administration. Blood analyses were performed by the Clinical Diagnostic Laboratories of the William R. Pritchard Veterinary Medical Teaching Hospital of the University of California, Davis, using standard protocols. The study was conducted in accordance with the Institutional Animal Care and Use Committee of the University of California at Davis and according to ARRIVE guidelines.
Experimental induction of inflammation/lameness
Synovial inflammation and forelimb lameness was induced in all sixteen horses using a previously described model of LPS induced synovitis [3, 5, 11]. Briefly, LPS (E.coli O55:B5; Sigma-Aldrich, St. Louis, MO) was prepared in a sterile manner, at a concentration of 100 ng/mL in Dulbecco PBS solution. The area surrounding the right antebrachiocarpal joint was clipped and prepared for injection and localized inflammation induced by sterile injection of 100 ng (1 mL) of LPS. Additional doses of LPS were administered in the same manner on days 4 and 9.
Instrumentation and drug administration
Prior to drug administration, 8 horses were randomly assigned to the control (saline) group and 8 horses to the treatment (isoflupredone acetate) group, using a random number generator. The control group was included for pharmacodynamic assessments. Horses were treated with saline or drug 12 h post-administration (Day 0) of the first LPS dose. For horses receiving isoflupredone acetate, prior to drug administration, a 14-gauge catheter was placed in one external jugular vein for collection of blood samples.
For drug or saline administration, the area over the right antebrachiocarpal joint was scrubbed with chlorhexidine solution and 70% isopropyl alcohol, the joint flexed and a total dose of 8 mg of isoflupredone acetate (Predef 2X, Zoetis, Florham Park, NJ) or an equivalent volume of saline (control group) was administered aseptically into the joint. The dose chosen for this study was based upon the previous study conducted by our laboratory .
For drug concentration determinations, blood samples were collected at time 0 and at 15, 30, and 45 min, and 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 18, 24, 36, 48 and 72 h post isoflupredone acetate administration. Catheters were removed following collection of the 24-h sample and the remaining samples collected by direct venipuncture. Blood samples were collected into EDTA blood tubes, centrifuged at 3000 × g and plasma immediately transferred into storage cryovials and stored at -20◦C until analysis.
Synovial fluid samples were collected from the right antebrachiocarpal and middle carpal joints by aspiration with a sterile needle prior to LPS administration, immediately prior to drug administration and at 24, 48, 72, 96 and 120 h and on day 7, 9, 10, 14, 21 and 28-days post-administration. Samples were collected from the left leg prior to drug administration and on days 4, 9 and 28 post drug administration. Synovial fluid was stored at -20 °C until analysis for determination of drug concentrations.
Urine samples were collected from all horses via free catch for measurement of isoflupredone concentrations. Samples were collected on Day 0 (prior to drug administration) and at 24, 48 and 72 h post drug administration. All samples were stored at -20◦C until analyzed for determination of isoflupredone concentrations.
Sample analysis (plasma/synovial fluid/urine drug concentrations)
The concentration of isoflupredone was measured in plasma, synovial fluid and urine by liquid chromatography-tandem mass spectrometry (LC–MS/MS) as described previously .
Pharmacokinetic modeling of isoflupredone concentration data
Isoflupredone plasma concentration data from a previous study, in which 12 horses received a single intra-articular 8 mg dose of isoflupredone acetate (Predef 2X, Zoetis, Florham Park, NJ) into a non-inflamed joint  were pooled with the concentration data generated from the current study. This additional data allowed for determination of the most appropriate pharmacokinetic model for simultaneous modeling of the plasma and synovial fluid concentration data. Incorporation of this data set also allowed for a comparison of data generated in the two studies. Synovial fluid samples used in the analysis were those collected at 24, 48, 72 and 96 h post-administration.
For data generated in the current study (LPS model), a non-compartmental analysis (NCA) was performed on the plasma concentrations as an aid in determining initial estimates for subsequent model fitting. A number of models were fit to the data (Supplemental Data), including one and two compartments for each of the synovial fluid (Csyn) and plasma concentrations (Cp). In addition, the effect of LPS on the parameters was assessed via modeling LPS as a covariate. The final model that was employed used a single compartment for each of Csyn and Cp. The absorption of drug from the synovial fluid to plasma was modeled as irreversible, and separate rates of appearance of drug in plasma (Kap) values were determined for LPS vs non-LPS data. The final model used a clearance parameterization, and relevant half-lives of interest were derived from the clearances and volumes of distribution. The Csyn and Cp data for all horses were modeled simultaneously using a nonlinear mixed modeling approach with the Phoenix NLME software program (V22.214.171.1240; Certara, Princeton, NJ).
Multiplicative residual error models were used for both Csyn and Cp data, and the effect of LPS on Kap was also modeled via an exponential function.
Pharmacodynamic assessments were determined immediately prior to LPS administration (-12 h prior to isoflupredone acetate or saline administration), time 0 (immediately prior to isoflupredone acetate or saline administration) and at 12, 24, 48, 72, 96 (immediately prior to LPS2) and 108 h and on days 5, 7, 9 (pre LPS3), 10, 10.5, 14, 17, 21, 24 and 28 post drug administration. All assessments were performed by an experienced board-certified equine surgeon (ACVS) blinded to treatment. For lameness evaluations, horses were required to walk and trot in a straight line and were scored according to guidelines established by the American Association of Equine Practitioners (AAEP) [12, 13]. The range of motion of the antebrachiocarpal joints was determined by passive flexion and the tendency to pull away. The joint was flexed and a goniometer positioned at the center of rotation of the joint was used to measure the maximal angle of flexion [5, 13]. Three consecutive measurements were taken, and the average reported. The degree of joint distension was measured using a tape to measure the circumference of the antebrachiocarpal joint.
Changes from baseline (time 0) were computed for each of the horses and t-tests were performed separately for each time post dosing to determine if any of the changes from baseline for circumference, flexion and lameness scores were statistically significant between the saline and isoflupredone treatment groups. A level of statistical significance of 0.05 was used. These tests should be considered as descriptive in nature and no adjustment was made for the multiplicity of tests that were run.