Pharmacokinetics of Ketorolac Tromethamine in Horses After Intravenous, Intramuscular, and Oral Single-Dose Administration

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J. vet. Pharmacol. Therap. 39, 167--175. doi: 10.1111/jvp.12260. Pharmacokinetics of ketorolac tromethamine in horses after intravenous, intramuscular, and oral single-dose administration

A. W. BIANCO* Bianco, A. W., Constable, P. D., Cooper, B. R., Taylor, S. D. Pharmacokinetics P. D. CONSTABLE† of ketorolac tromethamine in horses after intravenous, intramuscular, and ‡ oral single-dose administration. J. vet. Pharmacol. Therap. 39, 167–175. B. R. COOPER & S. D. TAYLOR* Nonsteroidal anti-inflammatory drugs (NSAIDs) are an integral component of equine analgesia, yet currently available NSAIDs are both limited in their *Department of Veterinary Clinical Sciences, analgesic efficacy and have adverse effects. The NSAID ketorolac trometha- College of Veterinary Medicine, Purdue mine (KT) is widely used in humans as a potent morphine-sparing analgesic University, West Lafayette, IN, USA; drug but has not been fully evaluated in horses. The purpose of this study †Department of Veterinary Clinical was to determine the pharmacokinetic profile of KT in horses after intra- Medicine, College of Veterinary Medicine, University of Illinois, Urbana-Champaign, venous (i.v.), intramuscular (i.m.), and oral (p.o.) administration. Nine IL, USA; ‡Bindley Bioscience Center, Purdue healthy adult horses received a single 0.5-mg/kg dose of KT via each route of University, West Lafayette, IN, USA administration. Plasma was collected up to 48 h postadministration and analyzed for KT concentration using HPLC/MS/MS. Noncompartmental analysis of i.v. dosage indicated a mean plasma clearance of 8.4 (mL/min)/kg and an estimated mean volume of distribution at steady-state of 0.77 L/kg. Noncom- partmental analysis of i.v., i.m., and p.o. dosages indicated mean residence times of 2.0, 2.6, and 7.1 h, respectively. The drug was rapidly absorbed after i.m. and p.o. administration, and mean bioavailability was 71% and 57% for i.m. and p.o. administration, respectively. Adverse effects were not observed after i.v., i.m., and p.o. administration. More studies are needed to evaluate the analgesic and anti-inflammatory properties of KT in horses. (Paper received 25 November 2014; accepted for publication 20 July 2015) Sandra D. Taylor, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA. E-mail: [email protected]

INTRODUCTION system based on physiological and behavioral parameters. In a retrospective study of 300 equine cases of surgical colic, Pain management is an active area of investigation in postoperative pain was reported in 32% of horses that received veterinary medicine. Managing pain in equine patients relies flunixin meglumine (0.25 mg/kg i.v.) every 8 h after recovery primarily on drug intervention, as other aspects of multimodal from anesthesia. Pain was the most common reason recorded therapy are often impractical or cost prohibitive. Nonsteroidal for euthanasia in these horses (Mair & Smith, 2005). Opiate anti-inflammatory drugs (NSAIDs) are an integral component drugs, such as morphine and butorphanol, have been used to of equine pain management as they demonstrate both anti- provide adjunctive analgesia in horses receiving NSAIDs inflammatory and analgesic effects. The currently available (Sellon et al., 2004); however, their use is associated with NSAIDs labeled for use in horses are used to treat a wide significant side effects in horses including behavior changes variety of conditions associated with varying degrees of pain. and gastrointestinal hypomotility that often limit their use in Despite standard analgesic therapy, horses may still experience high-risk patients, regardless of pain score (Kohn & Muir, moderate to severe pain associated with certain conditions 1988; Sellon et al., 2004; Boscan et al., 2006). such as acute laminitis or following surgery. In a recent evalu- Ketorolac tromethamine (KT) is a pyrrolizine carboxylic acid ation of 34 horses undergoing exploratory laparotomy, 65% of derivative NSAID and nonselective cyclooxygenase (COX) inhi- horses experienced moderate to severe pain following surgery bitor. While it is widely used in human medicine, primarily as despite ‘standard of care’ analgesic therapy (Graubner et al., a postoperative analgesic for moderate to severe pain, KT has 2011). The horses in the Graubner et al.’s study received not been thoroughly evaluated in horses (Rooks et al., 1982; flunixin meglumine (0.5 mg/kg i.v.) every 8 h and were Planborg et al., 1994; Ready et al., 1994; Blackburn et al., evaluated for pain using a multidimensional pain scoring 1995; Etches et al., 1995; O’Hara et al., 1997; Ferraresi et al.,

2014). Ketorolac tromethamine is commonly administered ical analysis (SBA) and had no history of NSAID administra- intravenously (i.v.) or as a constant rate infusion (CRI) in tion within 2 months prior to the start of the study. A humans, even though it is labeled for intramuscular (i.m.) and repeated Latin square design was used to ensure that each oral (p.o.) use (Ready et al., 1994; Blackburn et al., 1995; horse received each route of drug administration (i.v., i.m., Etches et al., 1995). Numerous studies in human medicine p.o.). Horses were randomly assigned a number (1 through 9) have evaluated KT as a morphine-sparing analgesic in and divided into groups of 3, and each trial was staggered over postoperative patients. The results of these studies indicated a the course of 3 days. The first route of administration was ran- 22–44% reduction in morphine consumption when patients domly assigned to each horse. The horses consisted of six were treated with morphine and ketorolac for the first 24 h mares and three geldings that ranged in age from 6 to following abdominal or orthopedic surgery vs. morphine alone 24 years, with a mean of 15 6 years. Five breeds were rep- (Blackburn et al., 1995; Etches et al., 1995; O’Hara et al., resented, including three Standardbreds, two Thoroughbreds, 1997). The adverse effects of KT in humans are similar to two Warmbloods, one Saddlebred, and one Quarter Horse. The those associated with all NSAIDs, including gastrointestinal horses weighed 460–650 kg and were weighed immediately ulceration and increased risk of bleeding, but the overall prior to the start of each trial to ensure accurate dosing. All incidence of adverse effects in postoperative patients is low (Elia horses gained weight over the course of the study, with a et al., 2005). Ketorolac tromethamine has been shown to be mean weight gain of 21.6 9.6 kg. This was attributed to equivalent in safety to the NSAIDs diclofenac and ketoprofen access to lush forage as the study took place between April in postoperative human patients (Forrest et al., 2002). In rats, and June when the horses were housed on green pasture. All KT has demonstrated an anti-inflammatory potency up to 50 procedures in this study were approved by the Institutional times that of phenylbutazone and antipyretic properties 20 Animal Care and Use Committee at Purdue University. times that of aspirin (Rooks et al., 1982). When administered orally in mice, KT was found to be more than 350 times as Drug administration potent as phenylbutazone with respect to analgesia (Rooks et al., 1982, 1985). Studies in mice and rats have indicated A 0.5-mg/kg dosage of KT was used for i.v., i.m. (30 mg/mL; that the relative analgesic potency of KT is even greater than Wockhardt USA Inc., Parsippany, NJ, USA), and p.o. (10 mg/ its anti-inflammatory potency (Rooks et al., 1982, 1985). tablet; Teva Pharmaceuticals, North Wales, PA, USA) adminis- We are aware of only two studies that have investigated the tration, with a 2-week washout period between each trial. The pharmacokinetic profile of KT in adult horses. A 1994 study 0.5-mg/kg dose of KT was equivalent to a KT dose of reported pharmacokinetic data after a single 300 mg/kg i.v. or 0.34 mg/kg. Injectable drug doses were rounded up to the i.m. dose of KT in six adult horses (Planborg et al., 1994), but nearest 0.1 mL; oral doses were rounded up to the nearest the study failed to account for individual horse variability as it whole tablet. The washout period was selected to exceed the was not a crossover design. A more recent study evaluated KT anticipated elimination half-time by at least 10 times. For the in six colts (0.5 mg/kg i.v.) undergoing castration (Ferraresi first 24 h of each trial, the horses were housed in box stalls et al., 2014). The colts in that study received several other with free access to grass hay, alfalfa hay, and water. The medications concurrently and underwent general anesthesia, horses were not fasted before or after p.o. administration to two factors that have the potential for confounding kinetic mimic clinical administration. After the first 24 h of each trial data as it is unknown how drug interactions or anesthesia and during the washout period, the horses were housed on affects metabolism of KT in the horse. The objective of the pasture. study reported here was therefore to evaluate the pharmacoki- Intravenous jugular catheters were aseptically placed the netic profile of KT in healthy adult horses after i.v., i.m., and night before the start of each trial; horses undergoing i.v. drug p.o. single-dose administration and to investigate the potential administration had a catheter placed in each jugular vein. The for adverse effects following KT administration. A single dosage catheter used to deliver the KT was removed after drug admin- of 0.5 mg/kg was selected for investigation based on extrapola- istration; the contralateral catheter was used for all blood tion from recommended human dosage protocols and previous collections. Intramuscular injection of KT was performed in the pharmacokinetic studies in the horse and dog (Planborg et al., neck muscle opposite that of the jugular catheter. The oral 1994; Mathews et al., 1996; Pasloske et al., 1999; Cagnardi dose of KT was delivered via nasogastric tube; the tablets were et al., 2013). first dissolved in 60 mL of water, added to the nasogastric tube, and then followed by 3 L of water to ensure complete delivery of the drug. MATERIALS AND METHODS Heparinized blood samples were collected from the jugular vein catheter immediately prior to drug administration (t = 0) Animals and experimental design and at 5, 10, 15, 20, 30, 45, 60, and 90 min and 2, 3, 4, 6, Nine adult horses from the Purdue University teaching herd 8, 10, 12, 24, and 48 h after drug administration. Plasma was were utilized for the crossover pharmacokinetic study. The harvested by centrifugation at 1300 g for 5 min within 6 h of horses were determined to be healthy on the basis of physical collection and stored at 80 °C until analyzed. Ketorolac examination, complete blood count (CBC), and serum biochem- tromethamine is stable in plasma for at least 30 days when

© 2015 John Wiley & Sons Ltd Ketorolac in horses 169 stored at 20 °C (Mroszczak et al., 1987) or 80 °C (Raju gas temperature = 350 °C and flow rate = 9 L/min, nebulizer et al., 2012). pressure = 40 psi, sheath gas temperature = 250 °C, sheath gas flow rate = 7 L/min, and capillary potential = 3500 V. All data were collected and analyzed with Agilent MassHunter Adverse effects B.03 software. Quantitation was based on a 6-point standard Horses were continually monitored during the first 12 h of curve, with KT concentrations ranging from 2.5 to 5000 ng/ each trial, and complete physical examinations were performed mL, using a diluent of acetonitrile and water (1:1, v/v). Stan- at t = 0, 4, 12, 24, and 48 h. A CBC (Abbott Cell-Dyn 3500 dard curves were fit to a quadratic function, with a 1/x curve Hematology Analyzer, Abbott Park, IL, USA) and SBA fit weighting. Correlation coefficients >0.9997 were obtained. (Johnson & Johnson Vitros 5,1 FS Chemistry Analyzer, Curves were used if the standard concentration accuracy for Holliston, MA, USA) were performed at t = 0 and t = 24 h. each point was between 95% and 105%. Responses for KT Subjective assessments were made regarding changes in behav- were normalized against the internal standard (RR = response ior, appetite, and fecal consistency, as well as evidence of ratio). inflammation at the i.m. injection site (characterized by the The limit of quantitation was 1.2 ng/mL and the limit of presence of heat, swelling, or pain). detection was 0.5 ng/mL, as defined as a signal-to-noise (SNR) ratio of 10:1 and 3:1, respectively, determined using authentic standards. Matrix effects and extraction recoveries were Sample analysis assessed using the approach detailed by Trufelli et al. (2011). Sample preparation. Plasma samples were prepared as described Matrix effects were determined by ratioing the RR in matrix- prior to high-performance liquid chromatography (HPLC) matched standards to the RR in neat standards. The matrix tandem mass spectrometry (MS-MS) analysis (Radwan et al., effect was determined at 2.5 (low), 250 (middle), and 5000 2010; Raju et al., 2012). Briefly, an internal standard (IS) (high) ng/mL, which were 122%, 127%, and 99.2%, respec- solution containing 50 lL etodolac (500 ng/mL in 50% tively (n = 6). Extraction efficiencies were determined by ratio- water:50% acetonitrile) was added to 200 lL plasma and ing the RR in preextraction spiked plasma to matrix-matched vortexed (Patrick et al., 2011). Protein precipitation was standards. The extraction efficiencies were determined at 2.5 performed by adding 800 lL of a solution of 0.1% formic acid (low), 250 (middle), and 5000 (high) ng/mL, which were in acetonitrile. The mixture was vortexed and then centrifuged 84.3%, 89.0%, and 123%, respectively (n = 6). As no signifi- at 12 000 g for 5 min. Aliquots of 500 lL were transferred to cant matrix effects or extraction efficiencies were observed, HPLC vials with 10 lL submitted for HPLC/MS-MS analysis. calibrants and control samples were prepared using a solvent of acetonitrile and water (1:1, v/v). At 2.5 (low), 250 (middle), HPLC/MS-MS analysis. Ketorolac tromethamine plasma levels and 5000 (high) ng/mL, the precision (relative standard devia- were quantitated by HPLC/MS-MS. Separation was performed tion (RSD)) of the standard curves was 7.0%, 3.7%, and 1.7%, on an Agilent Rapid Res 1200 HPLC system using an Agilent respectively (n = 6). Based on a 100 ng/mL control sample, Zorbax XDB-C18 (2.1 9 50 mm, 3.5 lm) column (Agilent the intraday assay precision RSD ranged from 3.6% to 13.1%, ZORBAX Eclipse XDB column; Agilent Technologies, Santa and the interday precision (n = 7) was 8.3%. The intraday

Clara, CA, USA). Mobile phase A was H2O with 0.1% formic accuracy RSD ranged from 95.8% to 103.9%, and the interday acid, and mobile phase B was acetonitrile (ACN) with 0.1% accuracy (n = 7) was 98.8%. formic acid. A linear gradient elution was used as follows: initial conditions 35% B; 0–8 min: gradient to 70% B; 8– Pharmacokinetic analysis 8.5 min: gradient to 90% B; and 8.5–9.5 min: gradient held 90% B. During compound elution, a flow rate of 0.4 mL/min Pharmacokinetics were characterized using standard compart- was used. Column re-equilibration was 9.5–10.5 min: gradient mental and noncompartmental methods and a software to 35% B and 10.5–13.5 min: gradient held 35% B. During program (PKSolver, doi:10:1016/j.cmpb.2010.01.007). A re-equilibration, flow rate was increased to 0.6 mL/min and variety of weighting schemes were examined, and the best column flow was diverted to waste. Retention time for KT was model fit (inverse of concentration squared) was determined 3.2 min and for etodolac was 6.9 min. using Akaike’s information criterion and the smallest sum of Analytes were quantified using MS/MS utilizing an Agilent squared residuals. 6460 triple quadrupole mass spectrometer with electrospray Compartmental pharmacokinetic variables were attempted to ionization (ESI) (Agilent 6460 Triple Quadrupole mass spec- be obtained for i.v., i.m., and p.o. data from each horse by trometer; Agilent Technologies). Quantitation was based on fitting the plasma KT concentration–time data to a two- and multiple reaction monitoring (MRM). For KT, ESI-positive mode three-compartment open model using various weighting was used with a transition of 256.1–104.9 and a collision schemes. Compartment models could not be satisfactorily fit to energy (CE) of 18 V. For etodolac, ESI-negative mode was used i.v. data for the majority of horses and to i.m. and p.o. data for with a transition of 286.1–212.1 and a CE of 20 V. Both any horse. Noncompartmental analysis for i.v., i.m., and p.o. compounds used a fragmentor energy of 125 V and a dwell data from each horse was fit using the last three or more data time of 300 ms. Source parameters were as follows: nitrogen points from the semilog plasma concentration–time curve to

© 2015 John Wiley & Sons Ltd 170 A. W. Bianco et al. calculate the elimination rate constant (kZ, slope of the termi- The plasma concentration–time relationship of KT following nal linear phase). The area under the curve (AUC∞) and the 0.5-mg/kg i.m. or p.o. dosing is depicted in Fig. 1, and relevant area under the ﬁrst moment curve (AUMC∞) were calculated pharmacokinetic variables are summarized in Table 1. The for each horse and treatment from the KT concentration–time mean observed time to maximum concentration (tmax) after relationship using the trapezoidal method, with the area from i.m. or p.o. administration was 25 and 19 min, respectively, the last time point extrapolated to inﬁnity using the last indicating rapid absorption. The observed peak mean plasma measured plasma KT concentration and kZ. The elimination concentrations (Cmax) after i.m. or p.o. administration were half-life was calculated as 0.693/kZ. Mean residence time 0.58 and 0.31 lg/mL, respectively. The MRT was 2.0 h for (MRT) was calculated from the ratio of AUMC∞ to AUC∞. i.v., 2.6 h for i.m., and 7.1 h for p.o. administration. Calcu-

Total plasma clearance (Clp) was calculated as dose/AUC∞. lated mean bioavailability (F) of KT after i.m. and p.o. delivery

The apparent steady-state volume of distribution (Vd(ss)) was was 71% and 57%, respectively. 2 calculated as dose 9 AUMC∞/AUC1. Mean absorption time (MAT) for i.m. or p.o. administration was calculated as Adverse effects MATIM = MRTIM – MRTIV or MRTPO – MRTIV. Bioavailability was determined by dividing the i.m. or p.o. AUC∞ by the i.v. There were no significant changes in the physical examination AUC∞ and multiplying by 100. or CBC of any horse during any trial period (Table 2). No subjective changes were noted in the behavior, appetite, or fecal consistency of any horse during any trial period. While none Statistical analysis of the i.m. injection sites showed any clinical evidence of pain Significance was set at P < 0.05. Data were expressed as or inflammation, plasma creatinine kinase activity was mean SD, and plasma concentration–time data was pre- increased at 24 h after i.m. injection (P = 0.012). The mean sented in a semi-logarithmic graph. A mixed model analysis of CK value preinjection was 136 34 IU/L vs. a postinjection variance (ANOVA) with repeated measures was used to evaluate CK value of 162 50 IU/L (Table 2). the CBC and SBA data in order to account for the crossover experimental design (SAS 9.3, SAS Inc, Cary, NC, USA). DISCUSSION

RESULTS Similar to other species, the pharmacokinetic proﬁle of KT after i.v. administration was characterized by a low volume of distri- Pharmacokinetics bution and rapid clearance from the plasma compartment The plasma concentration–time relationship of KT following a (Santos et al., 2001; Nagilla et al., 2009). The drug was single 0.5-mg/kg i.v. dose is depicted in Fig. 1, and relevant rapidly absorbed after i.m. and p.o. administration, and no pharmacokinetic variables are summarized in Table 1. Plasma adverse effects were observed after single-dose administration. KT concentration was below the limit of detection for all horses Mean total plasma clearance of KT was higher in the current and routes of administration at 48 h. Mean total plasma clear- study (8.4 mL/min/kg) than previously reported for horses by ance (Clp) was rapid at 8.4 (mL/min)/kg, and the estimated Planborg et al. (1994) (2.8 mL/min/kg) and Ferraresi et al. mean volume of distribution at steady-state (Vd(ss)) was (2014) (5.6 mL/min/kg). Differences in drug clearance after 0.77 L/kg. i.v. administration of a water-soluble drug such as KT are primarily due to species differences in the rate of hepatic KT metabolism or renal blood ﬂow, as KT is primarily metabolized by the liver to form glucuronide conjugates and excreted by the kidneys (Mroszczak et al., 1987). Species differences in urine pH may impact plasma clearance of acidic drugs such as KT, as alkaline urine has the potential to ion trap KT and thereby prevent reabsorption; however, this is considered an unlikely reason for the high clearance in horses, as KT has a pKa value of 3.5, which indicates effectively full dissociation even in acidic urine (pH = 5.5). Moreover, clearance of KT after i.v. administration is variable in ruminants that typically have an alkaline urine, being 12.4 (mL/min)/kg in adult sheep (Santos et al., 2001) and 8.8 (mL/min)/kg in adult goats (Nagilla et al., 2009), but only 0.8 (mL/min)/kg in calves (Nagilla et al., 2007). Differences in drug clearance may also Fig. 1. Semi-logarithmic graph of plasma ketorolac tromethamine concentration (mean SD) vs. time after intravenous, intramuscular, and be due to differences in plasma protein binding between oral administration of ketorolac tromethamine (0.5 mg/kg) to healthy species, as small changes in binding percentage for highly horses (n = 9). bound drugs such as KT can have a large impact on drug

Table 1. Noncompartmental pharmacokinetic parameters and body weight for ketorolac tromethamine in healthy adult horses (n = 9) following a single (0.5 mg/kg) intravenous, intramuscular, and oral dose

Horse

Variable 123456789Mean SD CV (%)

Intravenous Body weight (kg) 565 511 546 585 541 564 461 492 634 544 52 9

C0 (lg/mL) 3.48 1.46 1.06 3.50 3.65 2.77 5.84 1.73 3.78 3.03 1.47 49 kz (per h) 0.049 0.549 0.555 0.051 0.032 0.120 0.483 0.045 0.171 0.228 0.231 101 t1/2 (h) 14.1 1.3 1.2 13.5 21.9 5.8 1.4 15.4 4.1 8.7 7.6 87 AUC∞ (h 9 lg/mL) 0.984 0.437 0.204 0.824 0.697 0.824 0.515 0.628 0.804 0.657 0.240 37 2 AUMC∞ (h 9 lg/mL) 3.171 0.196 0.075 2.131 3.614 0.802 0.095 2.641 0.577 1.478 1.415 96 MRT (h) 3.2 0.4 0.4 2.6 5.2 1.0 0.2 4.2 0.7 2.0 1.9 94

Clp ({mL/min}/kg) 4.8 9.7 22.3 5.9 6.5 5.7 7.5 6.5 6.6 8.4 5.4 64 Vd(ss) (L/kg) 0.93 0.26 0.49 0.92 2.01 0.33 0.08 1.65 0.28 0.77 0.67 87 Intramuscular Body weight (kg) 554 504 578 563 529 579 460 493 650 546 57 10

Cmax (lg/mL) 0.31 0.70 0.67 0.52 1.55 0.39 0.37 0.40 0.29 0.58 0.39 68 tmax (h) 0.50 0.25 0.17 0.25 0.17 0.75 0.33 0.33 1.00 0.42 0.29 69 kz (per h) 0.238 0.057 0.080 0.430 0.204 0.055 0.065 0.178 0.060 0.152 0.126 83 t1/2 (h) 2.9 12.1 8.6 1.6 3.4 12.5 10.7 3.9 11.6 7.5 4.5 60 AUC∞ (h 9 lg/mL) 0.621 0.836 0.633 0.659 1.019 0.662 0.647 0.488 0.626 0.688 0.152 22 2 AUMC∞ (h 9 lg/mL) 1.160 2.766 1.354 0.828 1.257 2.313 3.429 0.676 2.212 1.777 0.944 53 MRT (h) 1.9 3.3 2.1 1.3 1.3 3.5 5.3 1.4 3.5 2.6 1.4 53 MAT (h) 1.4 2.9 1.8 1.3 3.9 2.5 5.1 2.8 2.8 0.6 3.1 489 F (%) 65 84 64 68 110 67 65 52 63 71 17 24 Oral Body weight (kg) 572 491 581 599 506 584 467 477 662 549 66 12

Cmax (lg/mL) 0.22 0.15 0.46 0.32 0.12 0.30 0.24 0.79 0.23 0.31 0.20 64 tmax (h) 0.25 0.33 0.25 0.25 0.50 0.17 0.17 0.50 0.33 0.31 0.13 41 kz (per h) 0.083 0.133 0.152 0.077 0.100 0.098 0.092 0.100 0.117 0.106 0.024 23 t1/2 (h) 8.3 5.2 4.6 9.0 7.0 7.1 7.5 6.9 5.9 6.8 1.4 21 AUC∞ (h 9 lg/mL) 0.576 0.313 0.775 0.562 0.611 0.424 0.379 0.931 0.342 0.546 0.208 38 2 AUMC∞ (h 9 lg/mL) 5.997 1.954 3.815 5.491 5.447 1.923 2.877 4.840 2.124 3.830 1.661 43 MRT (h) 10.4 6.2 4.9 9.8 8.9 4.5 7.6 5.2 6.2 7.1 2.2 31 MAT (h) 7.2 5.8 4.6 7.2 3.7 3.6 7.4 1.0 5.5 5.1 2.1 42 F (%) 60 31 78 58 66 43 38 99 35 57 22 40

C0, plasma concentration at time = 0h;Cmax, maximum plasma concentration; tmax, time to observed maximum plasma concentration; kz, elimination rate constant; t1/2, elimination half-life; AUC∞, area under the plasma concentration vs. time curve; AUMC∞, area under the ﬁrst moment curve; MRT, mean residence time; Clp, total plasma clearance; Vd(ss), volume of distribution at steady-state; MAT, mean absorption time; F, percent bioavailability. SD, standard deviation; CV, coefﬁcient of variation.

availability for hepatic metabolism or urinary excretion (Nag- core body temperature (Gelman, 1976; Gelman et al., 1984; illa et al., 2007). Additional studies to determine the rate of Wood & Wood, 1984). Given the rapid clearance of KT in the hepatic metabolism are indicated to confirm the supposition horse, the most useful application of the drug in horses is likely that rapid hepatic metabolism is the reason for the high to be as a short-term i.v. CRI or oral administration in horses plasma clearance of KT in horses. with normal gastrointestinal motility. While there are no reports It is not clear why Planborg et al. (1994) found KT to have a in the veterinary literature, CRI of NSAIDs has been described in much slower clearance than that reported in our study, but only human medicine for treatment of fever or pain due to surgery or 3 horses were used in that study to estimate clearance, and their cancer (Beattie et al., 1997; Moselli et al., 2010; Grimsby et al., assay was able to detect KT only up to 3 h post administration, 2012). When administered as a CRI in humans, KT has been which was probably too short a time interval to provide an shown to be safe and effective at providing analgesia and reduc- accurate estimate of clearance. The colts in the Ferraresi et al. ing the use of opiates (Burns et al., 1991; Myers & Trotman, study received acepromazine, detomidine, ketamine, and diaze- 1994). pam in addition to KT before undergoing general anesthesia with The mean Vd(ss) of KT in the current study (0.77 L/kg; isoflurane; clearance of KT is expected to be decreased in horses median 0.49 L/kg) was similar to that for other NSAIDs receiving these drugs because of drug-induced decreases in (Mroszczak et al., 1987) and was most likely due to a moderately cardiac output, mean arterial pressure, renal blood flow, and high degree of plasma protein binding. While not specifically

Table 2. Mean SD of complete blood count and serum biochemical analysis parameters for intravenous (i.v.), intramuscular (i.m.), and oral (p.o.) administration before (before) and 24 h after (after) administration of ketorolac tromethamine (0.5 mg/kg) in nine adult horses

i.v. before i.v. after i.m. before i.m. after p.o. before p.o. after

Variable Units Reference range Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Total plasma protein g/dL 5.7–8.1 6.9 0.4 7.0 0.3 6.8 0.3 6.9 0.3 6.9 0.5 7.0 0.4 RBC M/lL 6.0–12.0 8.2 0.9 7.8 0.9 8.4 0.9 8.3 1.0 8.2 0.8 7.9 0.8 Hematocrit % 35.0–50.0 40.7 4.0 38.8 3.7 41.9 4.1 40.9 4.1 40.6 3.5 39.3 2.7 HGB g/dL 11.0–19.0 13.7 1.3 13.1 1.4 14.2 1.5 13.9 1.6 13.7 1.2 13.3 0.9 MCV fL 35.0–55.0 49.9 2.2 50.0 2.2 49.8 2.0 49.5 1.6 49.8 2.2 49.9 2.3 MCHC g/dL 30.0–36.0 33.7 0.5 33.8 0.7 33.8 0.5 33.9 0.8 33.8 0.6 33.9 0.4 WBC K/lL 6.0–12.0 7.6 2.0 7.9 2.5 7.2 1.4 7.8 1.5 7.6 2.2 7.4 2.3 Neutrophil K/lL 3.0–7.0 5.0 2.3 5.2 2.7 4.5 1.2 4.9 1.4 5.0 1.9 4.7 1.6 Lymphocyte K/lL 1.5–5.5 2.3 0.6 2.4 0.9 2.5 0.9 2.5 0.5 2.3 0.7 2.3 1.0 Monocyte K/lL 0.05–0.80 0.2 0.2 0.2 0.1 0.2 0.1 0.3 0.2 0.1 0.1 0.2 0.1 Eosinophil K/lL 0.0–0.4 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Glucose mg/dL 73–124 98.3 6.2 98.8 9.6 96.6 7.0 95.1 4.8 96.8 10.0 98.8 9.7 BUN mg/dL 8.0–27 18.4 3.5 17.3 4.4 18.2 4.4 18.3 4.9 18.8 3.8 18.0 4.3 Creatinine mg/dL 0.6–1.8 1.0 0.1 1.0 0.2 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 Phosphorus mg/dL 2.0–5.7 3.9 0.4 3.8 0.6 3.8 0.5 3.7 0.8 3.6 0.5 3.7 0.5 Calcium mg/dL 10.7–13.4 12.2 0.8 12.1 0.7 11.0 3.5 12.2 0.4 12.2 0.6 12.3 0.5 Sodium mM 132–144 138.2 2.4 138.7 1.7 138.7 2.1 138.0 2.5 138.3 1.4 138.7 1.4 Potassium mM 2.7–4.8 3.5 0.7 3.5 0.4 3.6 0.6 3.5 0.7 3.5 0.5 3.4 0.6 Chloride mM 94–103 100.1 2.0 99.6 1.7 100.1 2.2 99.0 2.0 99.8 2.3 98.9 1.3 Carbon dioxide mM 23–31 29.3 1.4 29.8 2.0 29.7 1.8 29.6 1.6 29.7 1.9 29.9 2.0 Anion Gap mM 12.0–20 12.3 1.4 12.8 1.8 12.5 1.4 12.9 1.8 12.4 2.4 13.3 1.5 Total Protein g/dL 4.7–7.5 6.7 0.4 6.6 0.3 6.6 0.4 6.7 0.4 6.6 0.5 6.8 0.4 Albumin g/dL 2.5–3.8 3.0 0.2 3.0 0.1 3.0 0.1 3.0 0.2 3.0 0.2 3.0 0.2 Globulin g/dL 2.2–3.8 3.7 0.4 3.7 0.3 3.7 0.3 3.7 0.3 3.7 0.3 3.7 0.2 AST IU/L 206–810 379.9 101.8 368.2 75.0 363.2 73.2 376.3 69.5 370.9 48.3 369.9 43.9 ALP IU/L 109–331 153.8 74.5 156.2 76.6 144.2 49.4 148.3 48.9 152.1 72.2 156.2 77.1 GGT IU/L 12.0–46 31.7 9.4 32.3 9.7 31.7 8.9 32.8 8.1 32.7 10.3 33.3 10.3 Bilirubin mg/dL 0.10–2.6 1.5 0.3 1.7 0.4 1.3 0.4 1.7 0.6 1.4 0.3 1.7 0.5 Magnesium mg/dL 1.6–2.7 1.6 0.2 1.7 0.2 1.7 0.2 1.7 0.2 1.7 0.1 1.7 0.2 Creatinine Kinase IU/L 88–453 161.4 67.3 159.0 40.8 135.7 33.5 162.1 50.2 135.8 39.2 153.9 65.2

evaluated in the current study, Ferraresi et al. (2014) reported a Oral administration of KT resulted in a tmax of 19 min in plasma binding of 76% in horse plasma. Plasma binding of KT is horses, which is shorter than the tmax in dogs (51 min 72.0% in mice (Mroszczak et al., 1987), 92.1% in rats (Mroszc- (Pasloske et al., 1999) and humans (53 min (Jung et al., zak et al., 1987), 98.9% in dogs (Cagnardi et al., 2013), and 1988)). While the oral pharmacokinetics of KT have been 99.2% in humans (Mroszczak et al., 1987). evaluated in goats and calves, it is difficult to compare oral The mean plasma elimination half-life of KT in this study drug administration in ruminants vs. nonruminant animals was 8.7 h (median 5.8 h). For comparison, sheep have a given the vast difference in drug absorption resulting in much shorter elimination half-life (18 min) after i.v. adminis- delayed tmax (6.5 h in calves and 8.9 h in goats) (Nagilla et al., tration (Santos et al., 2001), whereas studies in dogs (Pasloske 2007, 2009). The differences in tmax between horses and dogs et al., 1999), calves (Nagilla et al., 2007), and the 1994 may be related to method of administration; the tablets in the study in horses (Planborg et al., 1994) reported an elimina- current study were dissolved in 60 mL of water prior to admin- tion half-life similar to that observed in humans of approxi- istration and then followed by 3 L of water, which may have mately 4–6h. facilitated gastric emptying and therefore a faster rate of deliv- The pharmacokinetic profile of KT after i.m. injection could ery to the small intestine. The oral formulation given to dogs not be adequately characterized using a compartmental was reported to be a capsule, which may have delayed drug model; consequently, noncompartmental analysis was per- absorption (Pasloske et al., 1999). Ketorolac tromethamine formed. Ketorolac tromethamine was rapidly absorbed after was administered via nasogastric tube in this study to ensure i.m. injection in horses, with a mean absorption time of the horse received the entire drug amount and permit accurate 36 min (95% confidence interval for mean absorption time of estimation of oral bioavailability; however, this method 5.5–6.7 h) that was similar to that reported in sheep of administration did not permit evaluation of the potential (11 min) (Santos et al., 2001) and humans (46 min) (Jung influence of buccal drug absorption. While in a clinical setting et al., 1988). KT would likely be given via oral dose syringe, given the mod-

© 2015 John Wiley & Sons Ltd Ketorolac in horses 173 erate oral bioavailability of KT found in this study, and all While no adverse effects were noted in the current study, other studies in a variety of species (Mroszczak et al., 1987; NSAID use in all species is associated with damage to the Pasloske et al., 1999; Nagilla et al., 2007), it is unlikely that renal medulla and gastrointestinal mucosa due to inhibition buccal absorption would have had an impact on the plasma of prostaglandin E2 and vasoconstriction. The safety of KT KT concentration–time profile. Per os administration of drugs is has been evaluated extensively in humans with mixed not usually accompanied by water, and delivery likely results results. While several studies report that the risk of adverse in partial loss of drug. Thus, intragastric administration of KT effects is not higher than other NSAIDs (Forrest et al., 2002; may have falsely increased oral bioavailability in this study. Elia et al., 2005), other studies have cited KT has having a Interestingly, the plasma concentration–time relationship after higher relative risk of upper gastrointestinal bleeding in com- oral KT administration appeared to be biphasic, with a slightly parison with other human NSAIDs (Castellsague et al., higher plasma KT concentration present than anticipated from 2012). The risk of adverse effects in humans has been approximately 4–12 h after administration. A more exagger- shown to be increased in certain patients with preexisting ated pattern has been observed in dogs administered KT intra- conditions such as renal or gastrointestinal disease, coagu- venously (Cagnardi et al., 2013), leading to speculation that lopathy, or those receiving concurrent administration of enterohepatic cycling of KT may have been present. other NSAIDs. Given these potential risks in the human pop- As reported in humans and other species (Mroszczak et al., ulation, use of KT in the United States is restricted to no 1987; Jung et al., 1988; Pasloske et al., 1999; Nagilla et al., more than 5 days of treatment (Reinhart, 2000). While no 2007, 2009), the mean bioavailability of KT after both i.m. adverse effects have been noted in any of the previous vet- and p.o. administration was moderate at 71% and 57%, erinary single-dose pharmacokinetic studies, only one study respectively. This is similar to the i.m. bioavailability previously has specifically evaluated the safety of multiple doses of KT. reported in horses (69%) (Planborg et al., 1994). In this study, no difference in adverse effects was found While the target plasma concentration of KT for effective between KT and flunixin meglumine in dogs after three analgesia in horses is not known, an EC50 of 0.37 lg/mL for doses of either drug administered 6 h apart (Mathews et al., plasma KT concentration has been calculated for humans 1996). Given the potentially severe adverse effects of NSAIDs using pharmacodynamic modeling (Mandema & Stanski, in horses, the safety of KT must be evaluated further. 1996). Interestingly, oral administration of KT failed to achieve The increase in CK values noted after i.m. injection may or a mean plasma KT concentration of 0.37 lg/mL, and i.m. may not be clinically relevant. Creatine kinase is a sensitive administration resulted in plasma KT concentration exceeding indicator of muscle damage; a small amount of muscle damage 0.37 lg/mL for <1h. can result in a detectable elevation in CK and does not neces- Despite the extensive literature on analgesic properties of sarily result in clinically detectable pain or inflammation (Le- KT in humans, veterinary studies are sparse. Only one anal- febvre et al., 1996). None of the horses experienced clinical gesic trial has been performed in horses, when six colts evidence of injection site reaction in this study or had a CK undergoing elective castration received KT preoperatively value that was outside of the reference range; however, the (0.5 mg/kg i.v.) (Ferraresi et al., 2014). All colts experienced postinjection samples were collected 24 h after injection and adequate analgesia, as determined by a visual analog score, CK activity peaks 6–12 h after muscle injury (Anderson, although there was no untreated control group and investiga- 1975). While the KT administered to horses in this study was tors were therefore not masked to treatment (Ferraresi et al., formulated for i.m. use in humans and is commonly used with- 2014). Several studies have evaluated the clinical efficacy of out significant adverse effects, the potential for muscle damage KT in providing postoperative analgesia in dogs. In a random- may become apparent with repeated or prolonged dosing in ized controlled trial, KT given at a dose of 0.5 mg/kg i.m. horses. was as effective as flunixin meglumine (1.0 mg/kg i.m.) and In conclusion, the pharmacokinetic profile indicates rapid superior to both butorphanol (0.4 mg/kg i.m.) and oxymor- absorption when KT is administered orally or by i.m. injection phone (0.05 mg/kg i.m.) in providing consistent postoperative in healthy adult horses with no obvious adverse effects after a analgesia (Mathews et al., 1996). Similar findings were single 0.5-mg/kg dosage. Further studies are necessary to eval- observed in a clinical trial during which 15 dogs received KT uate the analgesic and anti-inflammatory properties of a CRI of (0.5 mg/kg i.v.) for analgesia associated with elective castra- KT in the horse, as well as the drug’s safety profile when tion, although no control group was used in that study administered as a CRI. (Cagnardi et al., 2013). Given the diversity of individual pain sensitivity and the variety of painful medical conditions, deter- mining the effective therapeutic concentration of a drug is ACKNOWLEDGMENTS difficult, even when patients can verbally communicate their pain level. Furthermore, the target therapeutic concentration This project was supported by the state of Indiana and the of a drug is likely to vary among species. Therefore, the Purdue University College of Veterinary Medicine research appropriate dosage of a drug must be based on attaining the account funded by the total wager tax, and the Purdue plasma concentration that has been shown to be both University College of Veterinary Medicine Department of effective and safe. Veterinary Clinical Sciences Graduate Student Competitive

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