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