Patrick T. Colahan

scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints

With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

Pharmacokinetics of dexamethasone following intra‐articular, intravenous, intramuscular, and oral administration in horses and its effects on endogenous hydrocortisone

This study investigated and compared the pharmacokinetics of intra-articular (IA) administration of dexamethasone sodium phosphate (DSP) into three equine joints, femoropatellar (IAS), radiocarpal (IAC), and metacarpophalangeal (IAF), and the intramuscular (IM), oral (PO) and intravenous (IV) administrations. No significant differences in the pharmacokinetic estimates between the three joints were observed with the exception of maximum concentration (Cmax ) and time to maximum concentration (Tmax ). Median (range) Cmax for the IAC, IAF, and IAS were 16.9 (14.6-35.4), 23.4 (13.5-73.0), and 46.9 (24.0-72.1) ng/mL, respectively. The Tmax for IAC, IAF, and IAS were 1.0 (0.75-4.0), 0.62 (0.5-1.0), and 0.25 (0.08-0.25) h, respectively. Median (range) elimination half-lives for IA and IM administrations were 3.6 (3.0-4.6) h and 3.4 (2.9-3.7) h, respectively. A 3-compartment model was fitted to the plasma dexamethasone concentration-time curve following the IV administration of DSP; alpha, beta, and gamma half-lives were 0.03 (0.01-0.05), 1.8 (0.34-2.3), and 5.1 (3.3-5.6) h, respectively. Following the PO administration, the median absorption and elimination half-lives were 0.34 (0.29-1.6) and 3.4 (3.1-4.7) h, respectively. Endogenous hydrocortisone plasma concentrations declined from a baseline of 103.8 ± 29.1-3.1 ± 1.3 ng/mL at 20.0 ± 2.7 h following the administration of DSP and recovered to baseline values between 96 and 120 h for IV, IA, and IM administrations and at 72 h for the PO.

Perspectives on the use of gene therapy for chronic joint diseases.

Advances in molecular and cellular biology have identified a wide variety of proteins including targeted cytokine inhibitors, immunomodulatory proteins, cytotoxic mediators, angiogenesis inhibitors, and intracellular signalling molecules that could be of great benefit in the treatment of chronic joint diseases, such as osteo- and rheumatoid arthritis. Unfortunately, protein-based drugs are difficult to administer effectively. They have a high rate of turnover, requiring frequent readministration, and exposure in non-diseased tissue can lead to serious side effects. Gene transfer technologies offer methods to enhance the efficacy of protein-based therapies, enabling the body to produce these molecules locally at elevated levels for extended periods. The proof of concept of gene therapies for arthritis has been exhaustively demonstrated in multiple laboratories and in numerous animal models. This review attempts to condense these studies and to discuss the relative benefits and limitations of the methods proposed and to discuss the challenges toward translating these technologies into clinical realities.

The effect of sildenafil citrate administration on selected physiological parameters of exercising Thoroughbred horses.

REASONS FOR PERFORMING STUDY Sildenafil, a phosphodiesterase-5 inhibitor vasodilator, increases cGMP concentrations by inhibiting enzymatic degradation. Marketed to treat erectile dysfunction in men, it also reduces pulmonary arterial pressure (PAP). Because it reduces PAP, sildenafil may enhance performance and/or prevent exercise induced-pulmonary haemorrhage (EIPH). OBJECTIVE To determine if sildenafil citrate administration altered commonly measured indices of performance or reduced EIPH in exercised horses. METHODS Thirteen athletically conditioned Thoroughbred horses (2 mares and 11 geldings, age 3-12 years) were administered sildenafil citrate or placebo in 2 crossover design exercise testing studies. In a step-wise test to exhaustion, inspired/expired gas analysis, blood lactate, heart rate, runtime and bronchoalveolar lavage (BAL) cytology were measured. In a 13 m/s test to exhaustion, blood lactate, heart rate, runtime, BAL cytology and pulmonary arterial pressure were measured. Data were analysed with paired and unpaired t tests, one-way ANOVA and Tukeys pair-wise multiple comparison and Friedman repeated measure analysis of variance on ranks. RESULTS The administration of sildenafil did not alter mean inspired/expired gas measurements, plasma lactate concentrations or acute pulmonary haemorrhage in either exercise test or pulmonary arterial pressure measurement in the 13 m/s trial. Heart rates in both stress tests were significantly different at submaximal speeds and during the early recovery period. Run time was not affected by sildenafil administration in the step-wise trial (P = 0.622) or in the 13 m/s trial (P = 0.059). CONCLUSIONS Sildenafil did not alleviate pulmonary haemorrhage or enhance performance-related indices in these trials. Sildenafil administration altered cardiovascular adaptation to intense exercise as evidenced by altered heart rates at submaximal speeds and post exercise. The effect of these alterations on other performance perimeters was not evident.

Pharmacokinetics of glycopyrrolate following intravenous administration in the horse.

Glycopyrrolate, designated a class 3 substance by the Associa-tion of Racing Commissioners International, Inc., is regulated inracing horses because of its potential to affect performance.Although it has veterinary clinical applications by inhibitingparasympathetic activity, its use near race day is prohibited andpositive reports from postrace samples in the US are relativelycommon. Accordingly, the American Association of EquinePractitioners identified glycopyrrolate as a therapeutic substanceused by race track practitioners for legitimate therapeuticpurposes, and the Racing Medication and Testing Consortium(RMTC) has requested studies of the disposition of glycopyrrolateas part of its efforts to acquire reliable data upon which topropose thresholds and withdrawal time recommendations fortherapeutic substances used in racing horses.Glycopyrrolate, a quaternary ammonium salt and syntheticanti-cholinergic drug, exerts peripheral anti-muscarinic effectson the respiratory tract without imparting substantial effects onthe central nervous system (CNS) compared to other muscarinicantagonists such as atropine. Glycopyrrolate differs from theseother muscarinic antagonists because it penetrates the CNSpoorly due to its highly polar quaternary ammonium group andits permanent ionization at physiological pH compared to itsmore lipophilic congeners.Previous studies have investigated glycopyrrolate pharmaco-kinetics in humans to a limited extent (Pentilla et al., 2001).However, to our knowledge, pharmacokinetic studies of thisdrug in the horse have not been reported likely due to limitationsin sensitivity of the methods that are commonly used. Quanti-tative methods with limits of detection and quantification wellbelow those of previously reported methods have recently beendeveloped and validated through the RMTC research program.These validated methods (Rumpler et al., 2010b) demonstratenecessary sensitivity, accuracy, and precision to measure plasmaconcentrations sufficient to perform pharmacokinetic analysisthrough the 24-h time period after administration of clinicallyrelevant doses to horses. Such investigations could contribute tothe RMTC effort to establish a plasma threshold and torecommend a withdrawal time for this drug in race horses.Therefore, this study investigated the disposition of glycopyrro-late following intravenous administration of a 1-mg dose in thehorse.Eight, healthy, adult, Thoroughbred geldings, ranging in agefrom 5 to 10 years and weighing from 518 to 580 kg were usedin these studies. All study horses were housed in grass paddocksat the University of Florida, Veterinary Medical Center (Gaines-ville, FL), maintained on a diet of commercially available grainmixture, and had open access to water and hay at all times.Horses were subjected to treadmill exercise (3 days⁄week) beforeand throughout the duration of these studies. The experimentalprotocol was approved, and facilities were inspected by theUniversity of Florida Institutional Animal Care and Use Com-mittee.All horses were administered 1 mg (1.72–1.93 lg⁄kg) ofglycopyrrolate (glycopyronium bromide, American Regent, Inc.,Shirley, NY, USA) into the right jugular vein. Whole bloodsamples were collected from the left jugular vein via needlevenipuncture into partially evacuated tubes containing lithiumheparin. Blood samples were stored on ice until the plasma wasconcentrated by centrifugation (2500–3000 rpm or 776–1318 g)at4 C for 15 min. Harvesting of plasma took placewithin 1 h of sample collection, and 2–4-mL aliquots of plasmawere immediately frozen at )20 C and stored within 24 h at)80 C until analyzed. Collection times included a timepointbefore drug administration and 5, 10, 15, 20, 30, and 45 minand 1, 2, 3, 4, 6, 8, 24, 48, 72, 96, and 168 h after intravenousadministration. Specimens were collected from two of the horsesonly through 24 h after dosing.Plasma glycopyrrolate concentrations were determined usinga fully validated ultra-performance liquid chromatography andtandem mass spectrometry (MS⁄MS) method as previouslydescribed (Rumpler et al., 2010b) in accordance with US FDArecommended guidelines for bioanalytical methods. The methodis characterized by a lower limit of quantitation (LLOQ) of0.05 pg⁄mL of plasma.Nonlinear least squares regression analysis was performed onplasma glycopyrrolate concentration vs. time data and pharma-cokinetic parameters for all horses were estimated with bothnoncompartmental and compartmental analysis using Phoenix

Efficacy of Mechanical versus Non‐Mechanical Sterile Preoperative Skin Preparation With Chlorhexidine Gluconate 4% Solution

OBJECTIVE To compare the efficacy of sterile preoperative skin antisepsis using either a 5-minute mechanical preparation or 5-minute non-mechanical preparation with chlorhexidine gluconate 4% solution. STUDY DESIGN Matched design, ANOVA. ANIMALS Healthy adult Thoroughbred horses (n = 30). METHODS Each horse had both surgical preparation methods randomly assigned to identical sites on the left or right upper thigh. Prepared sites were sampled and cultured for bacteria after each preparation step. RESULTS Mechanical and non-mechanical preparation techniques significantly reduced bacteria isolated from surface swab samples compared with samples taken from unprepared skin and after the preliminary rough prepared skin (P < 0.05). No difference in the number of skin-associated bacteria was detected between the mechanical and non-mechanical sterile preoperative preparation techniques (P = 0.77). Ten species of bacteria were identified by 16s PCR after final skin preparation. CONCLUSIONS Pre-surgical skin preparation without repeated mechanical scrubbing using chlorhexidine gluconate 4% solution (total contact time, 225 seconds) is effective in reducing bacterial counts.

The pharmacokinetics of methocarbamol and guaifenesin after single intravenous and multiple‐dose oral administration of methocarbamol in the horse

A simple LC/MSMS method has been developed and fully validated to determine concentrations and characterize the concentration vs. time course of methocarbamol (MCBL) and guaifenesin (GGE) in plasma after a single intravenous dose and multiple oral dose administrations of MCBL to conditioned Thoroughbred horses. The plasma concentration-time profiles for MCBL after a single intravenous dose of 15 mg/kg of MCBL were best described by a three-compartment model. Mean extrapolated peak (C0 ) plasma concentrations were 23.2 (± 5.93) μg/mL. Terminal half-life, volume of distribution at steady-state, mean residence time, and systemic clearance were characterized by a median (range) of 2.96 (2.46-4.71) h, 1.05 (0.943-1.21) L/kg, 1.98 (1.45-2.51) h, and 8.99 (6.68-10.8) mL/min/kg, respectively. Oral dose of MCBL was characterized by a median (range) terminal half-life, mean transit time, mean absorption time, and apparent oral clearance of 2.89 (2.21-4.88) h, 2.67 (1.80-2.87) h, 0.410 (0.350-0.770) h, and 16.5 (13.0-20) mL/min/kg. Bioavailability of orally administered MCBL was characterized by a median (range) of 54.4 (43.2-72.8)%. Guaifenesin plasma concentrations were below the limit of detection in all samples collected after the single intravenous dose of MCBL whereas they were detected for up to 24 h after the last dose of the multiple-dose oral regimen. This difference may be attributed to first-pass metabolism of MCBL to GGE after oral administration and may provide a means of differentiating the two routes of administration.

An interlaboratory study of the pharmacokinetics of testosterone following intramuscular administration to Thoroughbred horses

Testosterone is an anabolic androgenic steroid (AAS) that is endogenously produced by both male and female horses that also has the potential for abuse when administered exogenously to race horses. To recommend appropriate withdrawal guidelines so that veterinarians can discontinue therapeutic use prior to competition, the pharmacokinetics and elimination of testosterone were investigated. An aqueous testosterone suspension was administered intramuscularly in the neck of Thoroughbred horses (n = 20). The disposition of testosterone from this formulation was characterized by an initial, rapid absorption phase followed by a much more variable secondary absorption phase. The median terminal half-life was 39 h. A second focus of this study was to compare the testosterone concentrations determined by two different laboratories using a percentage similarity model with a coefficient of variation of 16.5% showing good agreement between the two laboratories results. Based on the results of this study, a withdrawal period of 30 days for aqueous testosterone administered IM is recommended.

MAGNETIC RESONANCE IMAGING SCORING OF AN EXPERIMENTAL MODEL OF POST-TRAUMATIC OSTEOARTHRITIS IN THE EQUINE CARPUS

Magnetic resonance imaging (MRI) is the most sensitive imaging modality to detect the early changes of osteoarthritis. Currently, there is no quantifiable method to tract these pathological changes over time in the horse. The objective of this experimental study was to characterize the progression of MRI changes in an equine model of post-traumatic osteoarthritis using a semiquantitative scoring system for whole-organ evaluation of the middle carpal joint. On day 0, an osteochondral fragment was created in one middle carpal joint (OCI) and the contralateral joint (CON) was sham-operated in 10 horses. On day 14, study horses resumed exercise on a high-speed treadmill until the completion of the study (day 98). High-field MRI examinations were performed on days 0 (preosteochondral fragmentation), 14, and 98 and scored by three blinded observers using consensus agreement. Images were scored based on 15 independent articular features, and scores were compared between and within-groups. On days 14 and 98, OCI joints had significantly (P ≤ 0.05) higher whole-organ median scores (29.0 and 31.5, respectively), compared to CON joints (21.5 and 20.0, respectively). On day 14, OCI joints showed significant increases in high-signal bone lesion scores, and osteochondral fragment number and size. On day 98, high-signal bone lesion, low-signal bone lesion, osteophyte formation, cartilage signal abnormality, subchondral bone irregularity, joint effusion, and synovial thickening scores were significantly increased in OCI joints. Study results suggest that the MRI whole-organ scoring system reported here may be used to identify onset and progression of pathological changes following osteochondral injury.

Regulatory control of glycopyrrolate in performance horses using validated UHPLC/MS-MS methods.

We describe a validated, rapid, sensitive, and specific UHPLC-MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5-2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The intra and inter-batch imprecisions were <10% RSD and accuracy of the method ranged between 94 and 104%. Glycopyrrolate remained detectable in urine samples collected through 168 h after intravenous administration and through 24h after oral administration. Analytical method validation requirements for linearity, specificity, precision, accuracy, stability, dilution integrity, matrix effect, and ruggedness have been fulfilled. The urine method described in this report is simple and efficient and is the first reported method with sufficient sensitivity, accuracy, and precision to regulate the use of glycopyrrolate in urine samples collected more than one day after dosing of horses. Urine to plasma glycopyrrolate concentration ratios were calculated and were approximately 100:1 in samples collected from 24h through the end of sample collection.