J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12191

Pharmacokinetics and pharmacodynamics comparison between subcutaneous and intravenous butorphanol administration in horses L. CHIAVACCINI* A. K. CLAUDE* J. H. LEE † M. K. ROSS † R. E. MEYER* & V. C. LANGSTON* *Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA; † Department of Basic Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA

Chiavaccini, L., Claude, A. K., Lee, J. H., Ross, M. K., Meyer, R. E., Langston, V. C. Pharmacokinetics and pharmacodynamics comparison between subcutaneous and intravenous butorphanol administration in horses. J. vet. Pharmacol. Therap. doi:10.1111/jvp.12191. The study objective was to compare butorphanol pharmacokinetics and physiologic effects following intravenous and subcutaneous administration in horses. Ten adult horses received 0.1 mg/kg butorphanol by either intravenous or subcutaneous injections, in a randomized crossover design. Plasma concentrations of butorphanol were measured at predetermined time points using highly sensitive liquid chromatography–tandem mass spectrometry assay (LC-MS/MS). Demeanor and physiologic variables were recorded. Data were analyzed with multivariate mixed-effect model on ranks (P ≤ 0.05). For subcutaneous injection, absorption half-life and peak plasma concentration of butorphanol were 0.10  0.07 h and 88  37.4 ng/mL (mean  SD), respectively. Bioavailability was 87%. After intravenous injection, mean  SD butorphanol steady-state volume of distribution and clearance was 1.2  0.96 L/kg and 0.65  0.20 L/kg/h, respectively. Terminal half-lives for butorphanol were 2.31  1.74 h and 5.29  1.72 h after intravenous and subcutaneous administrations. Subcutaneous butorphanol reached and maintained target plasma concentrations >10 ng/mL for 2  0.87 h (Mean  SD), with less marked physiologic and behavioral effects compared to intravenous injection. Subcutaneous butorphanol administration is an acceptable alternative to the intravenous route in adult horses. (Paper received 7 May 2014; accepted for publication 31 October 2014) Ludovica Chiavaccini, Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, PO Box 6100, Mississippi State, MS 39762, USA. E-mail: [email protected]

INTRODUCTION Relief of pain is recognized as an essential aspect of animal welfare in all species, but equine analgesia still relies on anecdotal inferences and mostly depends on NSAIDs (Figueiredo et al., 2012). Μu-opioid receptor agonists commonly used in other mammals give inconsistent results in horses (Sanz et al., 2009; Figueiredo et al., 2012). Butorphanol, a synthetic centrally acting k-receptor agonist and l-receptor antagonist, has been shown to provide good visceral analgesia for 60 and 90 min after intravenous administration in horses and ponies (Kalpravidh et al., 1984a,b; Muir & Robertson, 1985; Sanchez & Robertson, 2014). More recent findings confirmed that butorphanol alone or associated with a tranquilizer has antinociceptive effects and reduces the use of volatile anesthetics, with improvement of cardiovascular stability and recoveries (Hofmeister et al., 2008; Love et al., 2009, 2012; Sanz et al., 2009; Caure et al., 2010), probably due to supraspinal nociceptive modulation © 2014 John Wiley & Sons Ltd

(Spadavecchia et al., 2007). Various degrees of ataxia, pacing, tachycardia, and reduction in intestinal motility have been reported after intravenous administration of butorphanol (Sellon et al., 2001; Knych et al., 2013). The evidence cited above has led researchers in the field to explore different solutions to maintain therapeutic plasma butorphanol concentrations while minimizing the adverse side effects. Sellon and her colleagues (Sellon et al., 2001, 2004) found that a constant rate intravenous infusion of butorphanol decreased cortisol concentrations and pain scores for 24 h after celiotomy in the horse, with no side effects. Further work showed no advantage of the intramuscular administration of butorphanol over a single intravenous dose in adult horses (Sellon et al., 2009). The objectives of this study were a) to describe and compare the pharmacokinetics of a 0.1 mg/kg dose of butorphanol following subcutaneous and intravenous administration in ten horses, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay and b) to score behavioral, cardiovascular, 1

2 L. Chiavaccini et al.

and gastrointestinal effects after administration and to correlate plasma concentration, the route of administration, and the time after administration with these effects. Our hypothesis was that after subcutaneous administration butorphanol would have similar pharmacokinetics compared to intravenous administration, without causing excitement or clinically significant cardiovascular and gastrointestinal side effects.

Sample collection

The study protocol was approved by the Institutional Animal Care and Use Committee at Mississippi State University (IACUC #12-077).

Horses were left free in a stall for the entire sampling period. For each butorphanol administration, blood samples (approximately 8–10 mL) were collected from the left jugular vein by aspiration through the catheter with a syringe at the following time points: prior to drug administration (baseline) and at 5, 10, 20, 30, 40, 50 min, 1, 2, 3, 4, 8, 12, 24, and 36 h after intravenous or subcutaneous injection. The catheter was removed after 12 h, and the last two samples were collected through direct venipuncture. Blood samples were immediately injected into a heparinized glass tube and stored on ice until centrifugation for collection of plasma. Plasma was stored at 80 °C until analysis.

Animals

Analysis of plasma concentrations of butorphanol

Ten healthy adult horses (four stallions and six mares) of different breeds (six QH, two Paints, one Gaited Horse and one Palomino) with an age between 3 and 15 years old and a body weight ranging between 350 and 560 kg were provided by the Mississippi Agricultural and Forestry Experiment Station. Horses were up to date with vaccinations and anthelmintic treatments, and they were determined to be healthy on the basis of physical exams, complete blood count, and large animal chemistry panels performed on the day of arrival. Horses were housed in individual stalls in a quite wing separated from the rest of the hospital, and they were acclimated 1 day prior to the beginning of the study; they were fed hay four times per day and received water ad libitum during the entire observation period.

The concentration of butorphanol was measured by LC-MS/ MS. Briefly, stock solutions of butorphanol and levorphanol (Sigma-Aldrich, St Louis, MO, USA) were prepared at 1 mg/mL in methanol and acetonitrile, respectively. Butorphanol working solutions were prepared by dilution of the stock solution with methanol to concentrations of 0.4, 4, 20, 100, 500, 2500, and 10 000 ng/mL. Plasma calibrators were prepared by adding the working standard solution into drug-free plasma to final concentrations of 0.01, 0.1, 0.5, 2.5, 12.5, 62.5, 250 ng/mL. Calibration curves and negative control samples were prepared fresh for each quantitative assay. Prior to analysis, 500 lL of plasma was fortified with 20 lL of 6.25 lM levorphanol internal standard (125 pmol). To precipitate the plasma proteins and extract the drug, 1.25 mL methanol was added to the plasma and samples were kept at 20 °C for 30 min. After centrifugation for 20 min at 4 °C (16 100 g), the supernatant was transferred into fresh tubes and 5 lL of 30% (v/v) glycerol in methanol was added prior to evaporation of solvents in an evacuated centrifuge (Savant speedvac; Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). One hundred microlitre of water: methanol (95:5 v/v) containing 0.1% acetic acid was added to the residue to reconstitute the sample, prior to filtration through a 0.22-m microcentrifuge filter. Samples were maintained at 10 °C in an autosampler until injection. Ten microlitre of the sample was injected onto the LC-electrospray ionization (ESI)-MS/MS; ACQUITY UPLCâ (Waters Corporation, Milford, MA, USA) interfaced with a Thermo-Electron Access Max triple-quadrupole mass spectrometer (Thermo Fisher Scientific Inc). Detection and quantitation were conducted using selective reaction monitoring (SRM) for butorphanol and the internal standard levorphanol. The technique was optimized to provide limit of detection (LOD) of 0.01 ng/mL and limit of quantification (LOQ) of 0.1 ng/mL. Concentration used for quality control (QC) samples was 62.5 ng/mL. The intra-assay coefficient of variation and interassay coefficient of variation were 5% and 15%, respectively.

MATERIAL AND METHODS

Instrumentation and drug administration Each horse was hand restrained; 2 inch 9 2 inch areas of skin above the left and right jugular furrow were clipped, surgically prepared and desensitized with 1 mL of lidocaine 2% (Lidocaine hydrochloride; LLC Phoenix Pharmaceutical INC., St. Joseph, MO, USA) injected subcutaneous on the site of catheter placement. A 14GA 9 5.25 inch polyurethane over-the-needle catheter (MILA International, Inc., Erlanger, KY, USA) was placed in the left jugular vein. An 18GA 9 2 inch Teflon catheter (SURFLOâ ETFE I.V. Catheters; Terumo, Somerset, NJ, USA) was also placed in the right jugular vein for administration of intravenous butorphanol and was removed following regime. At the beginning of the experiment, each horse received two injections: a single dose of 0.1 mg/kg of butorphanol and an equivalent volume of saline. Horses randomly received one of the two injections intravenously, followed by 5 mL of heparinized saline, and the other subcutaneously on the left side of the neck, above the supraspinatus muscle. After a wash-out period of 96 h, horses that received the butorphanol intravenously received it subcutaneously and vice versa. A single investigator (LC) blind to the treatment given was responsible of administering the drug, collecting the blood samples, and recording the observations.

Pharmacokinetic analysis © 2014 John Wiley & Sons Ltd

6 L. Chiavaccini et al.

Fig. 2. Heart rate with respect to time (in min) following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses. The middle box represents the middle 50% of scores for the population. The lower and higher hinges of the box indicates the 25th and 75th percentile respectively, or interquartile range (IQR), and the horizontal line represents the median of the distribution. The upper and lower whiskers represent values outside the middle 50%. Values outside the upper and lower whiskers are outliers. *Indicates statistical differences (P < 0.05) from baseline. a Indicates statistical difference between treatments at each time point.

Fig. 3. Respiratory rate in bpm with respect to time (in min) following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses. Data are expressed in median and IQR. See legend for Fig. 2 for explanation of box plots. *Indicates statistical differences (P < 0.05) from baseline.

Fig. 4. Gastrointestinal score with respect to butorphanol plasma concentrations following administration of 0.1 mg/kg butorphanol to nine adult horses. Data do not differentiate between intravenous or subcutaneous injections. The time of sampling in min is noted next to each point. All data are expressed as median.

the plasma concentration of 10 ng/mL, which is associated with analgesia (Sellon et al., 2009), was slightly longer after subcutaneous injection (2.0  0.87 h, Mean  SD) than after intravenous injection (1.54  0.74 h, Mean  SD). Butorphanol plasma concentrations best fit a two-compartment model after subcutaneous administration. In agreement with previous studies in humans (Gaver et al., 1980), in horses (Sellon et al., 2001, 2009; Knych et al., 2013) and in other species (Singh et al., 2011; Guzman et al., 2014), in our study butorphanol best fit a two-compartmental model after intravenous injection. The mean terminal half-life for butorphanol after intravenous administration was 2.3  1.74 h. This differs significantly from the 0.75 h (44 min) originally reported after intravenous administration of 0.1 mg/kg butorphanol to adult horses (Sellon et al., 2001), but it is in agreement with more recent studies reporting an elimination half-life of 7.8 and 5.9 h following intravenous administration of 0.08 mg/kg (Sellon et al., 2009) and of 0.1 mg/kg butorphanol (Knych et al., 2013), respectively. The more sensitive methodology used in more recent studies allowed the detection of plasma butorphanol over a © 2014 John Wiley & Sons Ltd

4 L. Chiavaccini et al. Table 3. Plasma concentrations (ng/mL) of butorphanol in horses following a 0.1 mg/kg dose intravenously Time (h) 0.08 0.17 0.33 0.50 0.67 0.83 1.00 2.00 3.00 4.00 8.00 12.00 24.00 36.00

Horse 1

Horse 2

Horse 3

Horse 4

Horse 5

Horse 6

Horse 7

Horse 8

Horse 9

223.5 115.8 115.5 79.8 61.0 55.0 33.5 12.0 5.9 3.2 0.4 0.3 0.1 0.0

301.3 269.4 156.4 73.2 49.5 43.0 20.3 5.4 1.8 1.0 0.1 0.1

151.7 113.6 113.6 40.7 28.4 27.2 15.9 11.5 5.8 2.4

358.2 270.6 171.9 129.0 116.4 90.5 80.3 43.3 11.9 5.7 0.5 0.4

183.6 168.2 95.8 91.4 66.0 50.9 41.2 10.8 3.6 1.1 0.1

387.5 164.2 91.4 51.5 26.3 12.1 8.6 5.3 2.5 2.0 1.7 1.6 1.0 0.2

323.7 193.9 116.9 75.6 38.1 22.2 14.3 6.5 3.1 2.9 2.2 1.1 1.3 0.3

448.6 275.1 138.7 120.3 87.4 61.6 50.4 8.6 2.1 1.3 0.3 0.2

209.5 134.7 60.3 64.9 45.2 24.7 12.9 5.4 2.8 2.4 1.4 0.8 0.2

Mean  SD 251 214.3 113.9 78.6 56.9 40.6 28.4 11.4 4.1 2.1 0.81 0.59 0.62 0.242

             

112 92.67 36.52 29.89 29.26 24.16 24.13 12.21 3.14 1.57 0.822 0.57 0.609 0.0184

Table 4. Plasma concentrations (ng/mL) of butorphanol in horses following a 0.1 mg/kg dose subcutaneously Time (h) 0.08 0.17 0.33 0.50 0.67 0.83 1.00 2.00 3.00 4.00 8.00 12.00 24.00 36.00

Horse 1

Horse 2

Horse 3

Horse 4

Horse 5

Horse 6

Horse 7

Horse 8

Horse 9

63.5 68.9 81.3 57.8 46.6 37.0 20.1 10.4 5.0 2.3 0.8 0.5 0.0

91.2 157.3 183.2 159.6 101.7 79.8 50.8 12.7 7.3 3.3 1.2 0.3 0.1 0.1

46.3 81.4 123.1 113.7 97.4 58.4 50.7 15.8 6.7 2.9 0.9 0.3 0.1 0.0

42.8 47.1 99.8 78.6 85.2 20.0 42.1 38.8 25.0 11.6 4.0 1.5 0.8 0.3

31.1 70.2 80.0 66.2 32.6 49.9 39.2 16.6 7.1 3.6 1.4 0.3 0.0 0.0

33.4 57.0 74.1 66.4 59.4 52.0 45.6 9.3 7.8 4.3 2.6 1.3 0.3 0.2

21.3 49.4 59.3 83.5 88.5 86.7 67.9 20.0 6.8 6.0 2.0 1.1 0.2 0.0

32.3 59.2 87.4 78.5 63.3 60.5 49.5 11.1 4.8 2.5 0.9 0.4 0.1 0.0

26.2 49.7 61.7 75.5 49.7 34.5 31.4 6.0 3.1 1.8 1.5 0.4 0.2 0.1

1000

ng/mL

100

10

1

0.1

0.01

0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36

h

Fig. 1. Mean and standard deviation of plasma butorphanol after intravenous (closed circle) and subcutaneous (open circle) administration at 0.1 mg/kg to nine adult horses.

concentration after intravenous injection was 94.9 ng/mL, which was below the lower limit of a CI 95% (178.4 ng/mL) and below the lower 2.5 percentile of the population

Mean  SD 43 71 94 87 69 53 44 16 8.2 4.3 1.7 0.66 0.2 0.09

             

22 34.2 38.5 31.6 24.6 21.3 13.5 9.6 6.48 3.02 1.05 0.466 0.237 0.095

(107.7 ng/mL). Because of this, we elected to remove that horse from the study as an outlier. Although it is difficult to speculate on the reasons this horse’s concentrations were different enough to necessitate removal in a blind study, our main suspicion was incorrect regime of the drug prior to the experiment. All intravenous injections were performed through a catheter that was immediately flushed with saline, so the possibility of extravascular injection was quite remote. The data from both the subcutaneous and intravenous doses were best fit by a two-compartment open model. Compartmental pharmacokinetic parameters are shown in Table 5. Table 6 contains the noncompartmental pharmacokinetic parameters. Although the model described each individual horse well, there was a large amount of variability between horses for all compartmental and noncompartmental parameters. After subcutaneous injection, butorphanol was well absorbed (F = 0.87); with an absorption t½, Cmax and Tmax of 0.1  0.07 h, 88  37.4 ng/mL and 0.33 h (0.17–0.67), respectively. The elimination half-life of butorphanol after subcutaneous administration was 5.29  1.72 h. Steady-state volume of distribution, systemic clearance, and terminal elimination © 2014 John Wiley & Sons Ltd

PK/PD of subcutaneous butorphanol in horses 5 Table 5. Mean  SD of compartmental pharmacokinetic parameters following administration of butorphanol as a single dose of 0.1 mg/kg intravenously (IV) and subcutaneously (SC) to 9 adult horses Parameter C1 (lg/mL) k1 (/h) Cz (lg/mL) kz (/h) Terminal t1/2 (h) ka (/h) Absorption t1/2 (h) k10 (/h) k12 (/h) k21 (/h) Vc (L/kg)

IV (2 compartment) 0.26 2.7 0.011 0.3 2.31

    

0.099 1.29 0.0154 0.226 1.74

1.98 0.59 0.4 0.41

   

0.69 0.619 0.361 0.165

SC (2 compartment) 0.7 1.66 0.0037 0.131 5.29 6.7 0.10 1.15 0.45 0.179

         

1.25 0.815 0.00241 0.0425 1.716 4.43 0.066 0.191 0.64 0.0631

C1, zero-time intercept of distribution slope in the compartmental model; Cz, zero-time intercept of terminal slope in compartmental model; k1, distribution rate constant; kz, terminal rate constant (elimination rate constant for i.v. data); terminal t1/2, half-life derived from rate constant of terminal phase as 0.693/mean_kz; ka, absorption rate constant; absorption t1/2, absorption half-life derived as 0.693/ mean_ka; k10, k12, and k21, first-order intercompartmental transfer rate constants; Vc, apparent volume of distribution of the central compartment. Table 6. Mean  SD of noncompartmental pharmacokinetic parameters following administration of butorphanol as a single dose of 0.1 mg/kg intravenously (IV) and subcutaneously (SC) to nine adult horses Parameter AUC0-∞ (lg
h/mL) F CL (L/kg
h) MRT0-∞ (h) Vss (L/kg) Cmax (ng/mL) Tmax (h) *

IV 0.167  0.0556 0.65  0.196 1.9  1.61 1.2  0.96

SC 0.139 0.87 0.78 2.7 2 88 0.33

 0.042  0.290  0.225  1.2  0.9  37.4 (0.17–0.67)

AUC0-∞, area under plasma concentration–time curve to infinity; F, bioavailability; CL, total body clearance; MRT0-∞, mean resident time; Vss, apparent volume of distribution at steady-state; Cmax, peak butorphanol plasma concentration; Tmax, time at Cmax. *Median (range).

half-life after intravenous injection were 1.2  0.96 L/kg, 0.65  0.20 L/kg/h, and 2.31  1.74 h, respectively. Physiological response and behavioral monitoring Heart rate was significantly higher when butorphanol plasma concentrations were ≥10 ng/mL (P = 0.002). When considering all variables in the model, HR significantly increased over time (P = 0.01), reaching a peak above baseline 50 min postinjection. Subcutaneous injection was associated with lower HR than intravenous injection in the first 8 h postinjection and at 36 h (P < 0.0001). Changes in HR over time are shown in Fig. 2. Plasma concentration >50 ng/mL was associated with significantly lower RR (P = 0.004). Independently of © 2014 John Wiley & Sons Ltd

the route of injection, horses steadily increased their RR over time (Fig. 3), reaching a statistically significant difference from baseline between 50 and 60 min, 3–4 h and 12 h postinjection (P < 0.0001). None of the horses received a behavioral score of 3 and only two horses were scored 0 (both after subcutaneous injection) (Table 1). Due to lack of variability, we decided to dichotomize this variable as excited vs. not excited. Six of nine horses showed signs of excitement (anxiety, pacing, shifting weight, or flicking ears without a stimulus applied) within 10 min after intravenous injection, for a duration ranging between 5 and 40 min. One horse appeared excited for 20 min after subcutaneous injection, but it was excited prior to injection as well. As extrapolated from the mixed-effect logistic regression model, with plasma butorphanol concentration >50 ng/mL horses were almost three times more likely to show signs of excitement (OR 2.9, P < 0.0001). The odds of excitement were significantly higher between 5 and 30 min postinjection (P < 0.0001), but horses were 1.5 times less likely (P = 0.025) to show signs of excitement after subcutaneous injection than after intravenous injection. There was significant negative association between gastrointestinal motility and plasma concentration of butorphanol (P < 0.0001) (Fig. 4). There was a delay between the maximal measured plasma concentration and the minimum recorded gastrointestinal motility score, with the minimal motility recorded when plasma butorphanol concentration decreased 30% of the maximum recorded plasma concentration. Gastrointestinal motility was significantly lower than baseline for 20 min after injection and rose above baseline after 4 h post injection, independently of the injection site (P < 0.0001) (Fig. 5). Between 3 and 4 h postinjection GI motility was significantly lower after subcutaneous injection than after intravenous injection (P < 0.0001). Determination of manure production All horses passed manure by 3 and 4 h after intravenous and subcutaneous injection, respectively (Fig. 6). The frequency of defecation was negatively associated with the plasma concentration of butorphanol (P < 0.0001). Defecation frequency and weight of manure following butorphanol administration are depicted in Figs 7 and 8. Independently of the injection site, horses passed fewer piles of manure per hour, compared to baseline, during the entire study period (P < 0.0001). When considering all variables in the model, following subcutaneous injection horses passed overall less manure per hour at each time point (P < 0.0001) and the fecal weight was reduced within 4 h (P = 0.011) and between 4 and 8 h (P = 0.005) compared to intravenous injection. None of the horses showed signs of discomfort or pain at any time during the study.

DISCUSSION When administered subcutaneously in adult horses, butorphanol was quickly and highly absorbed and the duration above

6 L. Chiavaccini et al.

Fig. 2. Heart rate with respect to time (in min) following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses. The middle box represents the middle 50% of scores for the population. The lower and higher hinges of the box indicates the 25th and 75th percentile respectively, or interquartile range (IQR), and the horizontal line represents the median of the distribution. The upper and lower whiskers represent values outside the middle 50%. Values outside the upper and lower whiskers are outliers. *Indicates statistical differences (P < 0.05) from baseline. a Indicates statistical difference between treatments at each time point.

Fig. 3. Respiratory rate in bpm with respect to time (in min) following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses. Data are expressed in median and IQR. See legend for Fig. 2 for explanation of box plots. *Indicates statistical differences (P < 0.05) from baseline.

Fig. 4. Gastrointestinal score with respect to butorphanol plasma concentrations following administration of 0.1 mg/kg butorphanol to nine adult horses. Data do not differentiate between intravenous or subcutaneous injections. The time of sampling in min is noted next to each point. All data are expressed as median.

the plasma concentration of 10 ng/mL, which is associated with analgesia (Sellon et al., 2009), was slightly longer after subcutaneous injection (2.0  0.87 h, Mean  SD) than after intravenous injection (1.54  0.74 h, Mean  SD). Butorphanol plasma concentrations best fit a two-compartment model after subcutaneous administration. In agreement with previous studies in humans (Gaver et al., 1980), in horses (Sellon et al., 2001, 2009; Knych et al., 2013) and in other species (Singh et al., 2011; Guzman et al., 2014), in our study butorphanol best fit a two-compartmental model after intravenous injection. The mean terminal half-life for butorphanol after intravenous administration was 2.3  1.74 h. This differs significantly from the 0.75 h (44 min) originally reported after intravenous administration of 0.1 mg/kg butorphanol to adult horses (Sellon et al., 2001), but it is in agreement with more recent studies reporting an elimination half-life of 7.8 and 5.9 h following intravenous administration of 0.08 mg/kg (Sellon et al., 2009) and of 0.1 mg/kg butorphanol (Knych et al., 2013), respectively. The more sensitive methodology used in more recent studies allowed the detection of plasma butorphanol over a © 2014 John Wiley & Sons Ltd

PK/PD of subcutaneous butorphanol in horses 7

Fig. 5. Gastrointestinal score (based on a 0–16 score, as described in Table 2) with respect to time (in min) following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses. Gastrointestinal motility was measured by auscultation over the four abdominal quadrants for 1 min each and scored as reported in Table 2. The score of the four quadrants were summed together getting a total score ranging from 0 to 16. Data are expressed in median and IQR. See legend for Fig. 2 for explanation of box plots. *Indicates statistical differences (P < 0.05) from baseline. a Indicates statistical difference between treatments at each time point.

Fig. 6. Time to passage of the first pile of manure (in hours) following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses.

longer period of time, likely accounting for the difference in elimination half-life observed. The terminal half-life of butorphanol after subcutaneous injection (5.29  1.72 h) did not differ significantly from the intravenous injection. After intravenous injection, butorphanol had a large volume of distribution (1.2  0.96 L/kg), in agreement with previous studies in adult horses (Sellon et al., 2001, 2009; Knych et al., 2013). However, clearance values were determined to be different in these studies. Sellon et al. reported clearance values ranging between 1.26  0.57 L/kg/h (Sellon et al., 2001) and 0.28  0.10 L/kg/h (Sellon et al., 2009), compared to our study and the study from Knych et al. (2013) that had similar clearance values (0.65  0.20 and 0.8  0.58 L/kg/h, respectively). Difference in clearance might again be explained by differences in assay sensitivity and frequency of sampling; clearance is inversely proportional to AUC, consequently with a more sensitive analytical analysis and longer terminal slope, the calculated clearance is slower. © 2014 John Wiley & Sons Ltd

Fig. 7. Number of defecations (indexed per hour) at consecutive time points, following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses. Data are expressed in median and IQR. See legend for Fig. 2 for explanation of box plots. *Indicates statistical differences (P < 0.05) from baseline. a Indicates statistical difference between treatments at each time point.

The type of drug, the animal species, age and weight, concurrent systemic diseases, physical activity, the route of administration, and site of injection are the most important biological factors affecting drugs bioavailability; and depending on the region of the body, subcutaneous bioavailability can be greater than intramuscular bioavailability (Marshall & Palmer, 1979). In contrast with previously reported bioavailabilities of 66% and 37% after intramuscular injection in neonates (Arguedas et al., 2008) and adult horses (Sellon et al., 2009), respectively, butorphanol was highly absorbed from the subcutaneous site of injection. Lower peak plasma concentrations (29 ng/mL), but similar rate of absorption (Tmax: 28 min), were observed after subcutaneous injection of 0.25 mg/kg of butorphanol in dogs (Pfeffer et al., 1980).

8 L. Chiavaccini et al.

Fig. 8. Wet weight of manure produced per kg of body weight per hour at consecutive time points, following intravenous and subcutaneous administration of 0.1 mg⁄kg of butorphanol to nine adult horses. The wet weight of manure produced in each time range was measured, indexed by body weight and plotted over the time frame. Data are expressed in median and IQR. See legend for Fig. 2 for explanation of box plots. *Indicates statistical differences (P < 0.05) from baseline. a Indicates statistical difference between treatments at each time point.

The cardiovascular effects of butorphanol after intravenous or intramuscular administration in horses and ponies are controversial. Robertson et al. reported an increase in heart rate for 1 h after administration of 0.2 mg/kg intramuscular butorphanol in ponies (Robertson et al., 1981). In agreement with this study, Knych et al. observed an increase in heart rate after intravenous administration of 0.1 mg/kg butorphanol in adult horses (Knych et al., 2013). Other studies failed to report any cardiovascular change following administration of intravenous and intramuscular doses ranging between 0.1 and 0.4 mg/kg (Robertson et al., 1981; Sellon et al., 2001, 2009). We observed that HR was directly associated with plasma concentration of butorphanol and that it was significantly higher than baseline within the first 50 min postinjection, independent of the injection site. However, after subcutaneous administration, HR was significantly lower than after intravenous administration, probably due to the lower plasma concentrations reached. In agreement with Knych et al. (2013), we found that the maximum change in HR was delayed with respect to butorphanol administration. Respiratory rate increased over time as previously reported (Sellon et al., 2009), but we found the increase to be independent of the route of injection. The barn where horses were stabled was not climate-controlled and an increase in environmental temperature could have been the cause of the steady increase in RR over time. In agreement with Sanchez et al. (2008), plasma concentrations >50 ng/mL were associated with lower respiratory rate than concentrations 50 ng/mL was associated with restlessness and excitation. Subcutaneous administration, reaching lower peak plasma concentrations, was less likely to cause excitation. Unlike previous studies (Kalpravidh et al., 1984a,b; Knych et al., 2013), we did not observe appreciable incidents of sedation or ataxia after either mode of injection. It is well recognized that opioids have an inhibitory effect on the gastrointestinal tract motility in the horse in vivo (Kohn & Muir, 1988; Sojka et al., 1988; Sutton et al., 2002; Boscan et al., 2006; Figueiredo et al., 2012); however, the role of mu and kappa opioid receptors on different tracts of the equine intestine is still controversial. Studies have suggested that butorphanol alone (Rutkowski et al., 1989) or combined with xylazine (Rutkowski et al., 1989; Merritt et al., 1998) can reduce spontaneous myoelectric activity of the duodenum, cecum, and colon in horses and ponies during the first hour postinjection. On the other hand, Menozzi et al. suggested that mu and kappa receptors have a negligible role on myogenic spontaneous activity of equine jejunum in vitro, but that selective kappa receptor agonists reduced electricalinduced contractions (Menozzi et al., 2012). In our study, gastrointestinal motility was inversely proportional to butorphanol plasma concentration, decreasing significantly from pretreatment values between 5 and 20 min after injection, independent of the injection site. The minimum auscultation score was slightly delayed with respect to maximum plasma concentration of the drug, in agreement with previous reports (Sellon et al., 2001), and is possibly related to drug distribution. All horses passed manure within 4 h postinjection. This observation is in contrast with the study by Knych et al. (2013), where seven of ten horses did not pass manure during the first 8 h after administration of 0.1 mg/kg intravenous butorphanol. However, horses in that study were fasted for 12 h prior to and following drug administration, whereas horses in our study were fed small amounts of grass hay four times per day through the study. While fecal weight was not associated with plasma butorphanol concentration, route of injection or time, the incidence of defecation was negatively associated with plasma concentration and horses produced less piles of manure per hour compared to baseline for 36 h after administration independent of the route of injection. Similarly, Sellon et al. (2001) observed that horses treated with 0.1 mg/kg intravenous butorphanol bolus produced significantly fewer fecal piles compared to the control group in the first 24 h postinjection. However, the number of fecal piles passed during the entire observation period was not significantly different between treated and control groups, as well as the gastrointestinal tract transit time, measured with polyethylene glycol 3500 fecal concentration vs. time curve (Sellon et al., 2001). © 2014 John Wiley & Sons Ltd

PK/PD of subcutaneous butorphanol in horses 9

In conclusion, butorphanol is rapidly absorbed after subcutaneous administration and shows high bioavailability, with duration of effective plasma concentration comparable to that of the intravascular injection, but with reduced systemic side effects. Therefore, the subcutaneous route might be a useful alternative route for butorphanol administration in adult horses.

ACKNOWLEDGMENTS The authors acknowledge Zoetisâ for providing the butorphanol. They want also to acknowledge Dr. Cate Mochal-King for technical support with horses’ management. The authors thank Dr. Robert Wills and Dr. Francisco Olea-Popelka for their assistance with the statistical analysis. This study was generously funded by CVM Office for Research and Graduate Studies (ORGS) Internal Grants Program at Mississippi State University and by Zoetis.

REFERENCES Arguedas, M.G., Hines, M.T., Papich, M.G., Farnsworth, K.D. & Sellon, D.C. (2008) Pharmacokinetics of butorphanol and evaluation of physiologic and behavioral effects after intravenous and intramuscular administration to neonatal foals. Journal of Veterinary Internal Medicine, 22, 1417–1426. Boscan, P., Van Hoogmoed, L.M., Farver, T.B. & Snyder, J.R. (2006) Evaluation of the effects of the opioid agonist morphine on gastrointestinal tract function in horses. American Journal of Veterinary Research, 67, 992–997. Caure, S., Cousty, M. & Tricaud, C. (2010) Effects of adding butorphanol to a balanced anaesthesia protocol during arthroscopic surgery in horses. The Veterinary Record, 166, 324–328. Conover, W.J. & Iman, R.L. (1981) Rank transformations as a bridge between parametric and nonparametric statistics. The American Statistician, 35, 124–129. Figueiredo, J.P., Muir, W.W. & Sams, R. (2012) Cardiorespiratory, gastrointestinal, and analgesic effects of morphine sulfate in conscious healthy horses. American Journal of Veterinary Research, 73, 799–808. Gaver, R.C., Vasiljev, M., Wong, H., Monkovic, I., Swigor, J.E., Van Harken, D.R. & Smyth, R.D. (1980) Disposition of parenteral butorphanol in man. Drug Metabolism and Disposition, 8, 230–235. Guzman, D.S., Drazenovich, T.L., KuKanich, B., Olsen, G.H., Willits, N.H. & Paul-Murphy, J.R. (2014) Evaluation of thermal antinociceptive effects and pharmacokinetics after intramuscular administration of butorphanol tartrate to American kestrels (Falco sparverius). American Journal of Veterinary Research, 75, 11–18. Hofmeister, E.H., Mackey, E.B. & Trim, C.M. (2008) Effect of butorphanol administration on cardiovascular parameters in isoflurane-anesthetized horses - a retrospective clinical evaluation. Veterinary Anaesthesia and Analgesia, 35, 38–44. Kalpravidh, M., Lumb, W.V., Wright, M. & Heath, R.B. (1984a) Analgesic effects of butorphanol in horses: dose-response studies. American Journal of Veterinary Research, 45, 211–216. Kalpravidh, M., Lumb, W.V., Wright, M. & Heath, R.B. (1984b) Effects of butorphanol, flunixin, levorphanol, morphine, and xylazine in ponies. American Journal of Veterinary Research, 45, 217–223. Knych, H.K., Casbeer, H.C., McKemie, D.S. & Arthur, R.M. (2013) Pharmacokinetics and pharmacodynamics of butorphanol following

© 2014 John Wiley & Sons Ltd

intravenous administration to the horse. Journal of Veterinary Pharmacology and Therapeutics, 36, 21–30. Kohn, C.W. & Muir, W.W. 3rd (1988) Selected aspects of the clinical pharmacology of visceral analgesics and gut motility modifying drugs in the horse. Journal of Veterinary Internal Medicine, 2, 85–91. Love, E.J., Taylor, P.M., Clark, C., Whay, H.R. & Murrell, J. (2009) Analgesic effect of butorphanol in ponies following castration. Equine Veterinary Journal, 41, 552–556. Love, E.J., Taylor, P.M., Murrell, J. & Whay, H.R. (2012) Effects of acepromazine, butorphanol and buprenorphine on thermal and mechanical nociceptive thresholds in horses. Equine Veterinary Journal, 44, 221–225. Marshall, A. & Palmer, G. (1979). Injection sites and drug bioavailability. In Injection Sites and Drug Bioavailability. Eds van Miert, A.S.J.P.A.M., Frens, J. & van der Kreek, F.W., pp. 54–60. Elsevier Scientific Pub. Co., Zeist, The Netherlands. Menozzi, A., Pozzoli, C., Zullian, C., Poli, E., Serventi, P. & Bertini, S. (2012) Inhibition of motility in isolated horse small intestine is mediated by kappa but not micro opioid receptors. Equine Veterinary Journal, 44, 368–370. Merritt, A.M., Burrow, J.A. & Hartless, C.S. (1998) Effect of xylazine, detomidine, and a combination of xylazine and butorphanol on equine duodenal motility. American Journal of Veterinary Research, 59, 619–623. Muir, W.W. & Robertson, J.T. (1985) Visceral analgesia: effects of xylazine, butorphanol, meperidine, and pentazocine in horses. American Journal of Veterinary Research, 46, 2081–2084. Nolan, A.M., Beasley, W., Reid, J. & Gray, G. (1994) Effects butorphanol on locomotor activity ponies. Journal of Veterinary Pharmacology and Therapeutics, 17, 323–326. Pfeffer, M., Smyth, R.D., Pittman, K.A. & Nardella, P.A. (1980) Pharmacokinetics of subcutaneous and intramuscular butorphanol in dogs. Journal of Pharmaceutical Sciences, 69, 801–803. Robertson, J.T., Muir, W.W. & Sams, R. (1981) Cardiopulmonary effects of butorphanol tartrate in horses. American Journal of Veterinary Research, 42, 41–44. Rutkowski, J.A., Ross, M.W. & Cullen, K. (1989) Effects of xylazine and/or butorphanol or neostigmine on myoelectric activity of the cecum and right ventral colon in female ponies. American Journal of Veterinary Research, 50, 1096–1101. Sanchez, L.C. & Robertson, S.A. (2014) Pain control in horses: what do we really know? Equine Veterinary Journal, 46, 517–523. Sanchez, L.C., Elfenbein, J.R. & Robertson, S.A. (2008) Effect of acepromazine, butorphanol, or N-butylscopolammonium bromide on visceral and somatic nociception and duodenal motility in conscious horses. American Journal of Veterinary Research, 69, 579– 585. Sanz, M.G., Sellon, D.C., Cary, J.A., Hines, M.T. & Farnsworth, K.D. (2009) Analgesic effects of butorphanol tartrate and phenylbutazone administered alone and in combination in young horses undergoing routine castration. Journal of the American Veterinary Medical Association, 235, 1194–1203. Sellon, D.C., Monroe, V.L., Roberts, M.C. & Papich, M.G. (2001) Pharmacokinetics and adverse effects of butorphanol administered by single intravenous injection or continuous intravenous infusion in horses. American Journal of Veterinary Research, 62, 183–189. Sellon, D.C., Roberts, M.C., Blikslager, A.T., Ulibarri, C. & Papich, M.G. (2004) Effects of continuous rate intravenous infusion of butorphanol on physiologic and outcome variables in horses after celiotomy. Journal of Veterinary Internal Medicine, 18, 555–563. Sellon, D.C., Papich, M.G., Palmer, L. & Remund, B. (2009) Pharmacokinetics of butorphanol in horses after intramuscular injection. Journal of Veterinary Pharmacology and Therapeutics, 32, 62–65.

10 L. Chiavaccini et al. Singh, P.M., Johnson, C., Gartrell, B., Mitchinson, S. & Chambers, P. (2011) Pharmacokinetics of butorphanol in broiler chickens. The Veterinary Record, 168, 588. Sojka, J.E., Adams, S.B., Lamar, C.H. & Eller, L.L. (1988) Effect of butorphanol, pentazocine, meperidine, or metoclopramide on intestinal motility in female ponies. American Journal of Veterinary Research, 49, 527–529. Spadavecchia, C., Arendt-Nielsen, L., Spadavecchia, L., Mosing, M., Auer, U. & van den Hoven, R. (2007) Effects of butorphanol on the

withdrawal reflex using threshold, suprathreshold and repeated subthreshold electrical stimuli in conscious horses. Veterinary Anaesthesia and Analgesia, 34, 48–58. Sutton, D.G., Preston, T., Christley, R.M., Cohen, N.D., Love, S. & Roussel, A.J. (2002) The effects of xylazine, detomidine, acepromazine and butorphanol on equine solid phase gastric emptying rate. Equine Veterinary Journal, 34, 486–492.

© 2014 John Wiley & Sons Ltd