J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12179
Pharmacokinetics and pharmacodynamics of dermorphin in the horse M. A. ROBINSON* , † F. GUAN* S. McDONNELL* C. E. UBOH* , † & L. R. SOMA* *Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, USA; †PA Equine Toxicology and Research Laboratory, West Chester, PA, USA
Robinson, M. A., Guan, F., McDonnell, S., Uboh, C. E., Soma, L. R. Pharmacokinetics and pharmacodynamics of dermorphin in the horse. J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12179. Dermorphin is a l-opioid receptor-binding peptide that causes both central and peripheral effects following intravenous administration to rats, dogs, and humans and has been identified in postrace horse samples. Ten horses were intravenously and/or intramuscularly administered dermorphin (9.3 1.0 lg/kg), and plasma concentration vs. time data were evaluated using compartmental and noncompartmental analyses. Data from intravenous administrations fit a 2-compartment model best with distribution and elimination half-lives (harmonic mean pseudo SD) of 0.09 0.02 and 0.76 0.22 h, respectively. Data from intramuscular administrations fit a noncompartmental model best with a terminal elimination half-life of 0.68 0.24 (h). Bioavailability following intramuscular administration was variable (47– 100%, n = 3). The percentage of dermorphin excreted in urine was 5.0 (3.7–10.6) %. Excitation accompanied by an increased heart rate followed intravenous administration only and subsided after 5 min. A plot of the mean change in heart rate vs. the plasma concentration of dermorphin fit a hyperbolic equation (simple Emax model), and an EC50 of 21.1 8.8 ng/mL was calculated. Dermorphin was detected in plasma for 12 h and in urine for 48 or 72 h following intravenous or intramuscular administration, respectively. (Paper received 24 October 2013; accepted for publication 18 September 2014) Mary A. Robinson VMD, PhD, PennVet Equine Pharmacology Laboratory, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348, USA. E-mail: [email protected]
INTRODUCTION Dermorphin (DER), originally isolated from the skin of the South American tree frog Phyllomedusa sauvagei, binds and activates the l-opioid receptor in mice, rats, guinea pigs, rabbits, dogs, squirrel monkeys, and humans (Broccardo et al., 1981; Sander & Giles, 1982; Sandrini et al., 1986; Melchiorri & Negri, 1996). Intravenous (i.v.) administration of DER (Dermorphin) to mice (Broccardo et al., 1981), dogs (Sander & Giles, 1982), and humans (Sandrini et al., 1986) has revealed both central and peripheral opioid-mediated effects even though only a small fraction (0.04% in rats) of DER crosses the blood–brain barrier (Negri & Improta, 1984). Effects on the cardiovascular, gastrointestinal, and neuromuscular systems have been demonstrated using in vitro and in vivo models (Broccardo et al., 1981; Sander & Giles, 1982; Sandrini et al., 1986). The first-known attempted use of DER as a therapeutic agent was by the Matses tribes of the upper Amazonian basin, who used the dried skin from Phyllomedusa frogs topically to treat © 2014 John Wiley & Sons Ltd
cuts during hunting expeditions (Negri et al., 2000). Possible effects noted were decreased pain sensation at the site of the cut and a transient excitation. These effects were not well documented and may not be attributed to DER alone as frog skin contains many other bioactive peptides (Negri et al., 2000). In ex vivo preparations, DER is 40 times more potent than morphine in reducing gastrointestinal motility (Broccardo et al., 1981). Following i.v. (Intravenous) administration to mice, DER is 11 times more potent than morphine in reducing nociceptive pain (Broccardo et al., 1981). The potency of DER and its analogs have stimulated interest in this group of peptides as a potential new class of analgesics. DER has not been studied in any clinical trials; however, analogs that more easily cross the blood–brain barrier and have longer lasting activity with fewer side effects are being studied (Broccardo et al., 2003; Schiller, 2005; Mizoguchi et al., 2011). Rumors of the illegal administration of DER to race horses prompted drug testing laboratories to develop analytical assays to detect DER in horse plasma and urine (Guan et al., 2013). Shortly after these methods were developed, DER was 1
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confirmed in postrace horse plasma and urine samples by 3 laboratories in the USA. The route, dose, and timing of DER administration, as well as the potential effects of the compound before and during the race, was unknown, and no published studies describing the effects of DER in horses were available. The goal of this study was to characterize the pharmacokinetics and pharmacodynamics of DER following i.v. or intramuscular (i.m.) administration to horses. MATERIALS AND METHODS Animals and experimental design Studies were conducted following protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee. All horses underwent routine dental and foot care and scheduled administration of appropriate vaccinations and deworming agents. The horses were no longer actively racing, were housed on pasture, and were not exercised on a routine basis. Based on physical examination and routine blood chemistry analysis, all the horses in the study were in good health. Horses were brought into temperature-controlled stalls 2 days before the study period. Studies were performed in June, August, and November. Stall temperature averaged 75°F during June and August and 60°F during November. The horses appeared comfortable at the initiation of the study. Grass hay and water were ad libitum. A total of six Thoroughbred and four Standardbred horses, comprising six females and fou geldings weighing 546.2 (468.2–653.0) kg, and ranging in age from 2 to 12 years were used in the study. Drug preparation and administration Dermorphin (ABBIOTEC, San Diego, CA, USA) powder was dissolved in HPLC-grade water for a final concentration of 1 mg/mL, and the solution was filtered by a 5-micron syringe filter prior to injection (Becton Dickinson Franklin Lakes, NJ, USA). Filtration was not expected to alter the final concentration of DER in the solution as complete dissolution occurred. A limitation of the study, however, is that the final concentration of DER following filtration was not verified. Two pilot studies were performed. A safety study was performed in a single horse to identify a dose with manageable behavioral effects. Serial boluses of 1, 1, 2, 3, and 3 mg (total of 10 mg) were administered directly into the jugular vein with each bolus given 5 min apart (total = 25 min). In another horse, 5 mg of DER was administered into the jugular vein at 0.54 lg/kg/min for 15 min. Subsequent studies evaluated the pharmacokinetics and pharmacodynamics of a bolus i.v. administration of DER into the jugular vein (5 mg within 10 sec, 9.5 1.1 lg/kg, n = 7) or an i.m. (Intramuscular) administration of DER into the neck (5 mg, 9.2 1.0 lg/kg, n = 6). Horses utilized in the pilot studies were included in subsequent studies following a minimum 4-week washout period. Three horses were administered DER by both routes (i.v. bolus and i.m.) with at least 4 weeks in between
treatment protocols. A total of 15 administrations to 10 horses were performed (2 i.v. pilots, 7 i.v. bolus, and 6 i.m.). Blood and urine collection An indwelling 14-g catheter (Angiocath, Becton Dickinson, Sandy, UT, USA) was placed into the contralateral jugular vein for collection of blood samples. Prior to placement, the jugular groove was clipped of hair, washed with sterile water and surgical soap, (Chlorhexidine gluconate, 4%, Purdue Products LP Stamford, CT, USA) and rinsed with a bactericide (Chlorhexidine diacetate, Fort Dodge Health, Fort Dodge, Iowa) and 70% isopropyl alcohol. Following the i.v. or i.m. administration of DER, blood samples were collected at 0, 2, 5, 10, 20, 30, and 45 min and at 1, 2, 3, 4, 6, 8, 10, 12 h postadministration. Plasma was harvested by centrifugation (2500–3000 revmin1, 776–1318 g) at 4 °C for 15 min. Centrifugation of blood took place within 15 min of sample collection and 3-mL aliquots of plasma were immediately frozen at 80 °C. Samples were only thawed once prior to quantification of DER. A sterile indwelling 24-F self-retaining catheter (Foley Catheters, CR Bard Inc., Covington, GA, USA) was placed in the bladder of female horses and attached to a drainage bag (Bard Center Entry Urinary Drainage Bag, CR Bard Inc.) for continuous collection of urine. Prior to placement, the vulva was washed with surgical soap and rinsed with sterile water. The total volume of urine excreted was collected and measured at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, and 24 h after drug administration. A sample was also collected by catheterization at 36, 48, and 72 h post-DER administration from some animals. Geldings were fitted with a urine collection bag for collection of urine at the same time points listed above. Aliquots of urine (3 mL each) were immediately frozen at 80 °C and only thawed once prior to quantification of DER. Quantification of DER The method used for the quantification of DER in plasma and urine samples was validated according to the FDA Guidance for Industry on Bioanalytical Method Validation (GFI #145, 2001) and is published (Guan et al., 2013). Briefly, equine plasma and urine samples (1 mL each) were treated with EDTA and urea, and then processed using solid-phase extraction (SPE). Resulting SPE (Solid phase extraction) eluates were dried under vacuum and analyzed by liquid chromatography hyphenated to tandem mass spectrometry (LTQ XL; ThermoFisher Scientific, San Jose, CA, USA) for DER. The method is validated for matrix effect, SPE efficiency, intraday and interday accuracy and precision, and stability of the analyte. With isotopically labeled DER as an internal standard (New England Peptide, Gardner MA), quantification linear range was 0.02– 10 ng/mL in plasma and 0.05–20 ng/mL in urine. The intraday and interday accuracy was from 91% to 100% for the low (0.05 ng/mL in plasma, 0.10 ng/mL in urine), intermediate (0.5 ng/mL in plasma, 1.0 ng/mL in urine), and high (5.0 ng/mL in plasma, 10.0 ng/mL in urine) concentrations. The © 2014 John Wiley & Sons Ltd
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intraday and interday coefficients of variation were 1 min apart), and the appearance of sweat was tabulated, and the time of the effect and return to baseline were recorded. Statistical analysis Comparison of selected pharmacokinetic parameters determined for the i.v. and i.m. routes was performed with a Wilcoxon
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nonparametric test (JMP Pro 10.0.0). Comparison of the duration of the latency period occurring prior to the observation of behavioral changes following i.v. and i.m. administration of DER was performed with an independent t-test (5.5 degrees of freedom) using a Satterhwaite correction for unequal variances. The effect of DER administration on HR was assessed by evaluating data from 30 min before to 60 min after administration (30 to 60 min) using a 2-way ANOVA and Dunnetts post hoc test. Control HR was designated as the HR obtained 1 min prior to DER administration (JMP Pro 10.0.0). RESULTS Pharmacokinetics
Table 1. Pharmacokinetic parameter estimates following a single intravenous (i.v.) or intramuscular (i.m.) administration of dermorphin to the horse (5 mg; i.v. = 9.5 1.1 lg/kg, n = 7; i.m. = 9.2 1.0 lg/kg, n = 6). Median and range are presented except for half-lives where harmonic mean pseudo SD was chosen (Lam et al., 1985) Parameter C0p , Cmax (ng/mL) AUC1 0 (hng/mL) Tmax (h) A (ng/mL) a (1/h) t1/2a (h) B (ng/mL) b (1/h) t1/2b (h) k (1/h) t1/2k (h)
i.v. 44.58 7.84 NA 44.05 7.43 0.09 0.69 0.90 0.76 NA NA
i.m.
(42.46–54.83) (5.58–11.04) (41.62–53.93) (5.20–11.06) 0.02 (0.51–1.71) (0.58–1.38) 0.22
5.25 4.78 0.50 NA NA NA NA NA NA 0.92 0.68
(4.93–6.72)* (3.8–8.03)* (0.33–0.75)
Plasma concentration vs. time data obtained following i.v. administration of DER was best described by a 2-compartment model (Fig. 1, Table 1). Dermorphin was distributed and eliminated rapidly from the plasma. At 2 min, the plasma concentration of DER was 54.8 15.3 ng/mL, declining to 27.1 3.9 ng/mL at 5 min, and 0.05 0.02 ng/mL at 3 h, which was the last time point at which DER concentration in all seven horses was above the lower limit of quantification (LLOQ) of 0.02 ng/mL. DER was below the LLOQ (Lower limit of quantification) in all seven horses at 8 h, but could still be detected in 5 of 7 horses at the last sampling time (12 h). Following i.m. administration, DER was absorbed quickly (Fig. 2). At 2 min, the plasma concentration of DER was 0.81 0.79 ng/mL, increasing to a Cmax of 5.45 0.66 ng/mL between 20 and 45 min, and declining to 0.10 0.12 ng/mL at 4 h, which was the last time point at which the plasma concentration of DER in all six horses was above the LLOQ of 0.02 ng/mL. DER plasma concentration was below the LLOQ in all horses at 10 h, and was detected in only 2 of 6 horses at the last sampling time (12 h). Bioavailability determined for the three horses in the cross-over experiments was 47, 58, and 100%.
The urine Cmax was 1016 425 ng/mL at 36 13 min following i.v. DER administration (n = 5, Fig. 3a). The concentration of DER in urine declined to 0.11 0.03 ng/mL by 24 h. Intravenously administered DER was detectable in urine at 48 h in 2/5 horses, but was below the LLOQ of 0.05 ng/mL and was no longer detectable by 72 h. The urine Cmax was 257 159 ng/mL at 2.2 1.5 h following i.m. DER administration (n = 6, Fig. 3b). The concentration of DER in urine declined to 0.06 0.03 ng/mL by 24 h. Intramuscularly administered DER was detectable in urine at 48 h in 5 of 6 horses, but was below the LLOQ of 0.05 ng/mL and was still
Fig. 1. Dermorphin (DER) plasma concentration vs. time observed (markers) and predicted (dotted lines) compartmental data following intravenous administration (n = 7). The horizontal line displays the lower limit of quantification (LLOQ) of 0.02 ng/mL.
Fig. 2. Dermorphin (DER) plasma concentration vs. time observed (markers) and predicted (dotted lines) noncompartmental data following intramuscular administration (n = 6). The horizontal line displays the lower limit of quantification (LLOQ) of 0.02 ng/mL.
(0.66–1.48) 0.24
C0p = predicted plasma concentration at time 0 following i.v. administration; Cmax = maximum measured plasma concentration following i.m. administration; AUC1 0 = area under the plasma concentration vs. time curve; Tmax = time at maximum measured plasma concentration; A and B = coefficients; a = rate of distribution; b = terminal rate of elimination; k = terminal rate of elimination via noncompartmental analysis; t1/2a, t1/2b, t1/2k = half-lives; NA = not applicable. *i.m. parameter was significantly different from the corresponding i.v. parameter (P < 0.05).
© 2014 John Wiley & Sons Ltd
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(a)
(a)
(b) (b)
Fig. 3. Dermorphin (DER) urine concentration vs. time data (markers) following intravenous (a) or intramuscular (b) administration. The horizontal line displays the lower limit of quantification (LLOQ) of 0.05 ng/mL.
detectable in the urine of 4 of 6 horses at 72 h, which was the last sample collected post-DER administration. The fe of DER was 5.0 (3.7–10.6) % (n = 5). Pharmacodynamics A 5-mg i.v. bolus of DER administered within 10 sec elicited a period of minor to moderate excitation with the most intense agitation beginning within an average of 21.0 2.7 sec of the injection and lasting for 20–90 sec. Three distinct types of behavioral changes were observed. The first type included specific head and neck responses consisting of head shaking/bobbing/nodding and muzzle flipping, abnormal head and neck postures, chewing, tongue extensions, lip grimaces, semi-yawns, increased blinking, furrowed brow, and ears back. The second type included tail swishing/smacking, leg lifts toward the belly, jerky tucking up of the abdominal muscles, sudden postural adjustments and activity, head turns back toward the abdomen, with ears turned back. The third type, when present, followed the first two types and consisted of a period of extreme quiet, as seen in horses under mild sedation. Six of the seven horses receiving DER by the i.v. route and 3 of the 6 horses receiving DER by the i.m. route displayed all three types of behavioral changes. Behavioral changes following i.m. administration of DER were conspicuously less intense, and the latency period from the time of injection to the initial changes in behavior was significantly longer than the latency period following i.v. administration of DER (67.3 12.1 vs. 21.0 2.7 sec, P < 0.05). © 2014 John Wiley & Sons Ltd
Fig. 4. Heart rate (HR) prior to and following i.v. (a) or i.m. (b) dermorphin administration. Arrow indicates time of administration (t = 0). Asterisk (*) indicates significantly different (P ≤ 0.0125) from HR at t = 1. Bpm = beats per min.
A significant increase in HR (P ≤ 0.0125) was observed from 2 to 5 min immediately post- i.v. administration of DER (Fig. 4a). No significant change in HR was observed during the 30-min control period prior to administration. Average HR 1 min prior to administration was 41 6 beats per min (bpm), and HR increased to a maximum of 63 27 bpm at 4 min postadministration. Fitting the mean change in HR (D HR) vs. the mean DER plasma concentration data to the simple Emax model yielded an Emax of 27 5 bpm and an EC50 of 21.1 8.8 ng/mL for this dose (Fig. 5). HR was not significantly affected by intramuscular administration of DER at the same dose (Fig. 4b).
Fig. 5. Absolute change in heart rate (D HR) vs. dermorphin (DER) plasma concentration (mean SEM) following intravenous DER administration. Sampling time is indicated in minutes (m). Predicted data generated using a simple Emax model are shown (solid line). Emax = 27 5 bpm, EC50 = 21.1 8.8 ng/mL.
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Respiratory rate increased by ≥20% over baseline in 4 of 7 horses that were administered DER by the i.v. route from 19 5 to 42 19 breaths per min and returned to baseline within 10 2 min. Intestinal borborygmi were decreased in 6/7 horses (sounds > 1 min apart) between 10 and 40 min post- i.v. administration of DER. Sweating was observed in 4 of 7 horses between 5 and 20 min post- i.v. administration of DER. Profuse sweating with dripping occurred in 2 of these horses, and DER was detected in the sweat collected via a test tube at 7 (0.79 ng/mL) or 13 (0.18 ng/mL) min post- i.v. administration of DER. All 4 horses were dry 1 h after the administration. No changes in rectal temperature were observed. When DER was administered via an i.v. infusion (5 mg at 0.5 lg/kg/min for 15 min), a slow i.v. bolus (5 mg at 8.3 lg/kg over ~1 min), or i.m. (5 mg at 9.2 1.0 lg/kg), changes in HR (Fig. 4b), RR, and sweating were not observed. However, behavioral changes were noted as described above, and intestinal borborygmi were briefly decreased (sounds > 1 min apart) at 12 2 min in 3 of 6 horses administered DER by the i.m. route.
DISCUSSION Combined pharmacokinetic (PK) and pharmacodynamic (PD) studies of DER were not available despite its administration to a number of species (Broccardo et al., 1981; Sander & Giles, 1982; Sandrini et al., 1986; Melchiorri & Negri, 1996). To our knowledge, this is the first combined PK and PD study of DER in a mammal and the first study to evaluate the i.m. route of administration. The presence of DER in postrace horse plasma and urine samples was confirmed by our laboratory with the same methodology utilized in this study (Guan et al., 2013) and prompted this investigation. Following i.v. administration, DER was distributed within a short period (min), and DER concentration was below the LLOQ in plasma within 8 h. Following i.m. administration, DER was rapidly absorbed, and DER concentration was below the LLOQ in plasma within 10 h. The plasma DER concentration measured following i.m. administration could not be fit to the same compartmental model structure in all animals: 4 of 6 animals appeared to fit a 2-compartment elimination model with a distribution phase similar to that observed when DER was intravenously administered, while only a single elimination phase was observed in 2 of 6 animals (models not shown). Thus, noncompartmental modeling was used to estimate the terminal elimination rate constant following i.m. DER administration (Table 1). Complete pharmacokinetic studies of DER using an analytical assay of comparable sensitivity are not available in other species for direct comparison with the results of the present study. Rapid distribution and elimination was observed in rats following i.v. administration with a reported distribution half-life of 1.3 0.4 min and with 95% purity, HPLC-grade water, filtration of the solution prior to administration, and the administration of no more than 5 mL as a bolus, which is 10 000-fold less than the average blood volume reported for horses (10.3 L/100 kg) (Marcilese et al., 1964) render effects due to the vehicle itself an unlikely possibility. The effects of DER appeared to be concentration dependent based on the observations that 1) the effects appeared with increasing dose during the pilot safety study and 2) most of these effects were not observed during a slow i.v. administration of DER (slow push or constant rate infusion) or during the i.m. administration of DER following which the Cmax was 11fold lower than the C0p after a fast i.v. bolus (Table 1). Many of these effects were consistent with those reported for other species administered DER, and similarities were noted between the effects of DER and those of morphine administration to the horse. Behavioral effects of DER in other species are variable and dose dependent, and occur presumably due to the binding of DER to central and/or peripheral l-opioid receptors (Erspamer, 1992; Melchiorri & Negri, 1996). In contrast to humans and monkeys, l-opioid receptor agonists, such as morphine, cause dose-dependent excitation in the horse (Combie et al., 1979). Thus, the excitation observed in horses during the present study was consistent with central activation of the l-opioid receptor by DER. Some of the behavioral changes (head and neck responses) were consistent with those observed as part of © 2014 John Wiley & Sons Ltd
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the well described equine head-shaking syndrome, which is thought to be due to trigeminal neuritis or neuralgia (Newton et al., 2000). Other behavioral changes (tail swishing/smacking, leg lifts toward the belly, head turns back toward the abdomen, with ears turned back) were consistent with colic. In addition, humans administered DER by the i.v. route reported feeling a peripheral tingling sensation, heaviness of limbs, oppression of the chest, and blurred vision (Sandrini et al., 1986). These subjective responses, which may indicate additional effects via binding to peripheral l-opioid receptors, could not be assessed in the horse, and thus, could not be ruled out as possible contributors to the observed behavioral and other physiological responses noted in the present study. Similar to the horses in this study, dogs administered DER (1 or 10 lg/kg) by the i.v. route respond with an acute increase in HR and blood pressure that begins ~30 sec after the bolus, reaches a maximum response at 90–120 sec, and returns to baseline by 5 min (Sander & Giles, 1982). Similarly, humans administered DER as a constant i.v. infusion (5.5 lg/kg/min) for 30 min also respond with statistically significant increases in HR and blood pressure (Degli Uberti et al., 1983), although one subject in a subsequent study had a hypotensive response (Sandrini et al., 1986). When morphine is administered intravenously to the horse, HR and blood pressure increase in a dose-dependent manner for 2 min (50 lg/kg) or 15 min (100 lg/kg or 120 lg/kg) (Muir et al., 1978; Figueiredo et al., 2012). Thus, the increased HR observed in the present study following the administration of DER by the i.v. route to the horse was consistent with DER’s activity as a l-opioid receptor agonist. The exact mechanism(s), however, responsible for the changes in HR observed following DER administration cannot be clearly delineated by the present study, and it is likely that effects of DER on multiple physiological systems contribute to the observed changes in HR. The mean change in HR was plotted against the mean DER plasma concentration and was observed to empirically fit the simple Emax model. This analysis was performed to illustrate the DER plasma concentration observed to elicit an effect on a sensitive physiological variable (HR) and is useful for comparison with plasma concentrations of DER measured in postcompetition samples. It is important to note, however, that there are several limitations to this analysis and that the maximal efficacy and potency of DER was not determined (i.e., the calculated Emax and EC50 are specific to the dose administered and the i.v. route of administration). First, as described above, a clear mechanism for the increased HR resulting from DER administration cannot be identified by this study, and it is likely multifactorial (CNS excitation and/or colic/peripheral effects). Although DER plasma concentration correlates with DHR, this study and analysis do not provide any information regarding the concentration of DER at the l-opioid receptors, the location of the receptors, and does not determine whether the effect on HR is due directly to receptor binding. Second, equilibrium between the undefined biophase and the plasma was not achieved due to the extremely rapid distribution and elimination of DER from © 2014 John Wiley & Sons Ltd
plasma when administered as an i.v. bolus. This means that the DHR observed at each DER plasma concentration may not be representative of the DHR that would be elicited if DER were administered as a constant rate infusion to maintain a designated steady-state plasma concentration. Third, the administration of a single dose does not enable determination of the true maximum effect DER is capable of having on HR (i.e., maximal efficacy was not measured). Larger doses are expected to further increase HR. However, the goal of this study was not to determine the maximal efficacy of DER, and the study of larger doses was not pursed due to safety concerns. Finally, the rapidity of the changes in DER plasma concentration and HR in comparison with the sampling rate do not adequately assess for the possibility of hysteresis. The documented effects of DER administration on RR in this study were more variable than the effects on HR. Morphine administration to the horse increases RR in parallel with the effects on HR and blood pressure (Muir et al., 1978; Figueiredo et al., 2012). As the effects of DER on HR were no longer significant after 5 min, and the first RR recorded in the present study was at ~5 min, the effects on RR may have been missed in some horses. Nostril flaring and agitation prior to 5 min were observed in the video footage of these administrations. The effects of l-opioid receptor agonists on intestinal motility are well described. DER is 40 times more potent than morphine at decreasing intestinal motility in an ex vivo guinea-pig ileum model (Broccardo et al., 1981). In the conscious horse, intestinal motility was easily assessed by auscultation of the abdominal cavity for borborygmi. Similar to the effects of morphine administration (Figueiredo et al., 2012), intestinal borborygmi were decreased (sounds > 1 min apart) following DER administration by the i.v. and i.m. routes in the majority of horses in the study, which was consistent with a direct effect of DER on the intestinal l-opioid receptor. The decrease in borborygmi and the behavioral signs observed suggest acute mild colic may have occurred due to DER administration. To our knowledge, sweating following i.v. administration of DER has not previously been reported in any species. However, flushing and sweating has been reported in some humans following morphine administration (Gutstein & Akil, 2001), and DER increases body temperature of normothermic rats (Broccardo, 1987). Dermorphin administration to the horse did not increase rectal temperature; however, significant sweating was observed in some horses. The presence of DER in paired plasma and urine samples obtained from several horses during routine postrace sample collection was confirmed by our laboratory and 2 others within the USA. All plasma concentrations measured by our laboratory were between 0.005 and 0.020 ng/mL. Although the dose and route of DER administration to the racehorses is not known, the presence of DER in both matrices and at detectable concentrations using the same analytical method as used for this study indicates that the administration (if by the i.v. or i.m. route) was
Pharmacokinetics and pharmacodynamics of dermorphin in the horse M. A. ROBINSON* , † F. GUAN* S. McDONNELL* C. E. UBOH* , † & L. R. SOMA* *Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, USA; †PA Equine Toxicology and Research Laboratory, West Chester, PA, USA
Robinson, M. A., Guan, F., McDonnell, S., Uboh, C. E., Soma, L. R. Pharmacokinetics and pharmacodynamics of dermorphin in the horse. J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12179. Dermorphin is a l-opioid receptor-binding peptide that causes both central and peripheral effects following intravenous administration to rats, dogs, and humans and has been identified in postrace horse samples. Ten horses were intravenously and/or intramuscularly administered dermorphin (9.3 1.0 lg/kg), and plasma concentration vs. time data were evaluated using compartmental and noncompartmental analyses. Data from intravenous administrations fit a 2-compartment model best with distribution and elimination half-lives (harmonic mean pseudo SD) of 0.09 0.02 and 0.76 0.22 h, respectively. Data from intramuscular administrations fit a noncompartmental model best with a terminal elimination half-life of 0.68 0.24 (h). Bioavailability following intramuscular administration was variable (47– 100%, n = 3). The percentage of dermorphin excreted in urine was 5.0 (3.7–10.6) %. Excitation accompanied by an increased heart rate followed intravenous administration only and subsided after 5 min. A plot of the mean change in heart rate vs. the plasma concentration of dermorphin fit a hyperbolic equation (simple Emax model), and an EC50 of 21.1 8.8 ng/mL was calculated. Dermorphin was detected in plasma for 12 h and in urine for 48 or 72 h following intravenous or intramuscular administration, respectively. (Paper received 24 October 2013; accepted for publication 18 September 2014) Mary A. Robinson VMD, PhD, PennVet Equine Pharmacology Laboratory, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348, USA. E-mail: [email protected]
INTRODUCTION Dermorphin (DER), originally isolated from the skin of the South American tree frog Phyllomedusa sauvagei, binds and activates the l-opioid receptor in mice, rats, guinea pigs, rabbits, dogs, squirrel monkeys, and humans (Broccardo et al., 1981; Sander & Giles, 1982; Sandrini et al., 1986; Melchiorri & Negri, 1996). Intravenous (i.v.) administration of DER (Dermorphin) to mice (Broccardo et al., 1981), dogs (Sander & Giles, 1982), and humans (Sandrini et al., 1986) has revealed both central and peripheral opioid-mediated effects even though only a small fraction (0.04% in rats) of DER crosses the blood–brain barrier (Negri & Improta, 1984). Effects on the cardiovascular, gastrointestinal, and neuromuscular systems have been demonstrated using in vitro and in vivo models (Broccardo et al., 1981; Sander & Giles, 1982; Sandrini et al., 1986). The first-known attempted use of DER as a therapeutic agent was by the Matses tribes of the upper Amazonian basin, who used the dried skin from Phyllomedusa frogs topically to treat © 2014 John Wiley & Sons Ltd
cuts during hunting expeditions (Negri et al., 2000). Possible effects noted were decreased pain sensation at the site of the cut and a transient excitation. These effects were not well documented and may not be attributed to DER alone as frog skin contains many other bioactive peptides (Negri et al., 2000). In ex vivo preparations, DER is 40 times more potent than morphine in reducing gastrointestinal motility (Broccardo et al., 1981). Following i.v. (Intravenous) administration to mice, DER is 11 times more potent than morphine in reducing nociceptive pain (Broccardo et al., 1981). The potency of DER and its analogs have stimulated interest in this group of peptides as a potential new class of analgesics. DER has not been studied in any clinical trials; however, analogs that more easily cross the blood–brain barrier and have longer lasting activity with fewer side effects are being studied (Broccardo et al., 2003; Schiller, 2005; Mizoguchi et al., 2011). Rumors of the illegal administration of DER to race horses prompted drug testing laboratories to develop analytical assays to detect DER in horse plasma and urine (Guan et al., 2013). Shortly after these methods were developed, DER was 1
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confirmed in postrace horse plasma and urine samples by 3 laboratories in the USA. The route, dose, and timing of DER administration, as well as the potential effects of the compound before and during the race, was unknown, and no published studies describing the effects of DER in horses were available. The goal of this study was to characterize the pharmacokinetics and pharmacodynamics of DER following i.v. or intramuscular (i.m.) administration to horses. MATERIALS AND METHODS Animals and experimental design Studies were conducted following protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee. All horses underwent routine dental and foot care and scheduled administration of appropriate vaccinations and deworming agents. The horses were no longer actively racing, were housed on pasture, and were not exercised on a routine basis. Based on physical examination and routine blood chemistry analysis, all the horses in the study were in good health. Horses were brought into temperature-controlled stalls 2 days before the study period. Studies were performed in June, August, and November. Stall temperature averaged 75°F during June and August and 60°F during November. The horses appeared comfortable at the initiation of the study. Grass hay and water were ad libitum. A total of six Thoroughbred and four Standardbred horses, comprising six females and fou geldings weighing 546.2 (468.2–653.0) kg, and ranging in age from 2 to 12 years were used in the study. Drug preparation and administration Dermorphin (ABBIOTEC, San Diego, CA, USA) powder was dissolved in HPLC-grade water for a final concentration of 1 mg/mL, and the solution was filtered by a 5-micron syringe filter prior to injection (Becton Dickinson Franklin Lakes, NJ, USA). Filtration was not expected to alter the final concentration of DER in the solution as complete dissolution occurred. A limitation of the study, however, is that the final concentration of DER following filtration was not verified. Two pilot studies were performed. A safety study was performed in a single horse to identify a dose with manageable behavioral effects. Serial boluses of 1, 1, 2, 3, and 3 mg (total of 10 mg) were administered directly into the jugular vein with each bolus given 5 min apart (total = 25 min). In another horse, 5 mg of DER was administered into the jugular vein at 0.54 lg/kg/min for 15 min. Subsequent studies evaluated the pharmacokinetics and pharmacodynamics of a bolus i.v. administration of DER into the jugular vein (5 mg within 10 sec, 9.5 1.1 lg/kg, n = 7) or an i.m. (Intramuscular) administration of DER into the neck (5 mg, 9.2 1.0 lg/kg, n = 6). Horses utilized in the pilot studies were included in subsequent studies following a minimum 4-week washout period. Three horses were administered DER by both routes (i.v. bolus and i.m.) with at least 4 weeks in between
treatment protocols. A total of 15 administrations to 10 horses were performed (2 i.v. pilots, 7 i.v. bolus, and 6 i.m.). Blood and urine collection An indwelling 14-g catheter (Angiocath, Becton Dickinson, Sandy, UT, USA) was placed into the contralateral jugular vein for collection of blood samples. Prior to placement, the jugular groove was clipped of hair, washed with sterile water and surgical soap, (Chlorhexidine gluconate, 4%, Purdue Products LP Stamford, CT, USA) and rinsed with a bactericide (Chlorhexidine diacetate, Fort Dodge Health, Fort Dodge, Iowa) and 70% isopropyl alcohol. Following the i.v. or i.m. administration of DER, blood samples were collected at 0, 2, 5, 10, 20, 30, and 45 min and at 1, 2, 3, 4, 6, 8, 10, 12 h postadministration. Plasma was harvested by centrifugation (2500–3000 revmin1, 776–1318 g) at 4 °C for 15 min. Centrifugation of blood took place within 15 min of sample collection and 3-mL aliquots of plasma were immediately frozen at 80 °C. Samples were only thawed once prior to quantification of DER. A sterile indwelling 24-F self-retaining catheter (Foley Catheters, CR Bard Inc., Covington, GA, USA) was placed in the bladder of female horses and attached to a drainage bag (Bard Center Entry Urinary Drainage Bag, CR Bard Inc.) for continuous collection of urine. Prior to placement, the vulva was washed with surgical soap and rinsed with sterile water. The total volume of urine excreted was collected and measured at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, and 24 h after drug administration. A sample was also collected by catheterization at 36, 48, and 72 h post-DER administration from some animals. Geldings were fitted with a urine collection bag for collection of urine at the same time points listed above. Aliquots of urine (3 mL each) were immediately frozen at 80 °C and only thawed once prior to quantification of DER. Quantification of DER The method used for the quantification of DER in plasma and urine samples was validated according to the FDA Guidance for Industry on Bioanalytical Method Validation (GFI #145, 2001) and is published (Guan et al., 2013). Briefly, equine plasma and urine samples (1 mL each) were treated with EDTA and urea, and then processed using solid-phase extraction (SPE). Resulting SPE (Solid phase extraction) eluates were dried under vacuum and analyzed by liquid chromatography hyphenated to tandem mass spectrometry (LTQ XL; ThermoFisher Scientific, San Jose, CA, USA) for DER. The method is validated for matrix effect, SPE efficiency, intraday and interday accuracy and precision, and stability of the analyte. With isotopically labeled DER as an internal standard (New England Peptide, Gardner MA), quantification linear range was 0.02– 10 ng/mL in plasma and 0.05–20 ng/mL in urine. The intraday and interday accuracy was from 91% to 100% for the low (0.05 ng/mL in plasma, 0.10 ng/mL in urine), intermediate (0.5 ng/mL in plasma, 1.0 ng/mL in urine), and high (5.0 ng/mL in plasma, 10.0 ng/mL in urine) concentrations. The © 2014 John Wiley & Sons Ltd
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intraday and interday coefficients of variation were 1 min apart), and the appearance of sweat was tabulated, and the time of the effect and return to baseline were recorded. Statistical analysis Comparison of selected pharmacokinetic parameters determined for the i.v. and i.m. routes was performed with a Wilcoxon
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nonparametric test (JMP Pro 10.0.0). Comparison of the duration of the latency period occurring prior to the observation of behavioral changes following i.v. and i.m. administration of DER was performed with an independent t-test (5.5 degrees of freedom) using a Satterhwaite correction for unequal variances. The effect of DER administration on HR was assessed by evaluating data from 30 min before to 60 min after administration (30 to 60 min) using a 2-way ANOVA and Dunnetts post hoc test. Control HR was designated as the HR obtained 1 min prior to DER administration (JMP Pro 10.0.0). RESULTS Pharmacokinetics
Table 1. Pharmacokinetic parameter estimates following a single intravenous (i.v.) or intramuscular (i.m.) administration of dermorphin to the horse (5 mg; i.v. = 9.5 1.1 lg/kg, n = 7; i.m. = 9.2 1.0 lg/kg, n = 6). Median and range are presented except for half-lives where harmonic mean pseudo SD was chosen (Lam et al., 1985) Parameter C0p , Cmax (ng/mL) AUC1 0 (hng/mL) Tmax (h) A (ng/mL) a (1/h) t1/2a (h) B (ng/mL) b (1/h) t1/2b (h) k (1/h) t1/2k (h)
i.v. 44.58 7.84 NA 44.05 7.43 0.09 0.69 0.90 0.76 NA NA
i.m.
(42.46–54.83) (5.58–11.04) (41.62–53.93) (5.20–11.06) 0.02 (0.51–1.71) (0.58–1.38) 0.22
5.25 4.78 0.50 NA NA NA NA NA NA 0.92 0.68
(4.93–6.72)* (3.8–8.03)* (0.33–0.75)
Plasma concentration vs. time data obtained following i.v. administration of DER was best described by a 2-compartment model (Fig. 1, Table 1). Dermorphin was distributed and eliminated rapidly from the plasma. At 2 min, the plasma concentration of DER was 54.8 15.3 ng/mL, declining to 27.1 3.9 ng/mL at 5 min, and 0.05 0.02 ng/mL at 3 h, which was the last time point at which DER concentration in all seven horses was above the lower limit of quantification (LLOQ) of 0.02 ng/mL. DER was below the LLOQ (Lower limit of quantification) in all seven horses at 8 h, but could still be detected in 5 of 7 horses at the last sampling time (12 h). Following i.m. administration, DER was absorbed quickly (Fig. 2). At 2 min, the plasma concentration of DER was 0.81 0.79 ng/mL, increasing to a Cmax of 5.45 0.66 ng/mL between 20 and 45 min, and declining to 0.10 0.12 ng/mL at 4 h, which was the last time point at which the plasma concentration of DER in all six horses was above the LLOQ of 0.02 ng/mL. DER plasma concentration was below the LLOQ in all horses at 10 h, and was detected in only 2 of 6 horses at the last sampling time (12 h). Bioavailability determined for the three horses in the cross-over experiments was 47, 58, and 100%.
The urine Cmax was 1016 425 ng/mL at 36 13 min following i.v. DER administration (n = 5, Fig. 3a). The concentration of DER in urine declined to 0.11 0.03 ng/mL by 24 h. Intravenously administered DER was detectable in urine at 48 h in 2/5 horses, but was below the LLOQ of 0.05 ng/mL and was no longer detectable by 72 h. The urine Cmax was 257 159 ng/mL at 2.2 1.5 h following i.m. DER administration (n = 6, Fig. 3b). The concentration of DER in urine declined to 0.06 0.03 ng/mL by 24 h. Intramuscularly administered DER was detectable in urine at 48 h in 5 of 6 horses, but was below the LLOQ of 0.05 ng/mL and was still
Fig. 1. Dermorphin (DER) plasma concentration vs. time observed (markers) and predicted (dotted lines) compartmental data following intravenous administration (n = 7). The horizontal line displays the lower limit of quantification (LLOQ) of 0.02 ng/mL.
Fig. 2. Dermorphin (DER) plasma concentration vs. time observed (markers) and predicted (dotted lines) noncompartmental data following intramuscular administration (n = 6). The horizontal line displays the lower limit of quantification (LLOQ) of 0.02 ng/mL.
(0.66–1.48) 0.24
C0p = predicted plasma concentration at time 0 following i.v. administration; Cmax = maximum measured plasma concentration following i.m. administration; AUC1 0 = area under the plasma concentration vs. time curve; Tmax = time at maximum measured plasma concentration; A and B = coefficients; a = rate of distribution; b = terminal rate of elimination; k = terminal rate of elimination via noncompartmental analysis; t1/2a, t1/2b, t1/2k = half-lives; NA = not applicable. *i.m. parameter was significantly different from the corresponding i.v. parameter (P < 0.05).
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(a)
(a)
(b) (b)
Fig. 3. Dermorphin (DER) urine concentration vs. time data (markers) following intravenous (a) or intramuscular (b) administration. The horizontal line displays the lower limit of quantification (LLOQ) of 0.05 ng/mL.
detectable in the urine of 4 of 6 horses at 72 h, which was the last sample collected post-DER administration. The fe of DER was 5.0 (3.7–10.6) % (n = 5). Pharmacodynamics A 5-mg i.v. bolus of DER administered within 10 sec elicited a period of minor to moderate excitation with the most intense agitation beginning within an average of 21.0 2.7 sec of the injection and lasting for 20–90 sec. Three distinct types of behavioral changes were observed. The first type included specific head and neck responses consisting of head shaking/bobbing/nodding and muzzle flipping, abnormal head and neck postures, chewing, tongue extensions, lip grimaces, semi-yawns, increased blinking, furrowed brow, and ears back. The second type included tail swishing/smacking, leg lifts toward the belly, jerky tucking up of the abdominal muscles, sudden postural adjustments and activity, head turns back toward the abdomen, with ears turned back. The third type, when present, followed the first two types and consisted of a period of extreme quiet, as seen in horses under mild sedation. Six of the seven horses receiving DER by the i.v. route and 3 of the 6 horses receiving DER by the i.m. route displayed all three types of behavioral changes. Behavioral changes following i.m. administration of DER were conspicuously less intense, and the latency period from the time of injection to the initial changes in behavior was significantly longer than the latency period following i.v. administration of DER (67.3 12.1 vs. 21.0 2.7 sec, P < 0.05). © 2014 John Wiley & Sons Ltd
Fig. 4. Heart rate (HR) prior to and following i.v. (a) or i.m. (b) dermorphin administration. Arrow indicates time of administration (t = 0). Asterisk (*) indicates significantly different (P ≤ 0.0125) from HR at t = 1. Bpm = beats per min.
A significant increase in HR (P ≤ 0.0125) was observed from 2 to 5 min immediately post- i.v. administration of DER (Fig. 4a). No significant change in HR was observed during the 30-min control period prior to administration. Average HR 1 min prior to administration was 41 6 beats per min (bpm), and HR increased to a maximum of 63 27 bpm at 4 min postadministration. Fitting the mean change in HR (D HR) vs. the mean DER plasma concentration data to the simple Emax model yielded an Emax of 27 5 bpm and an EC50 of 21.1 8.8 ng/mL for this dose (Fig. 5). HR was not significantly affected by intramuscular administration of DER at the same dose (Fig. 4b).
Fig. 5. Absolute change in heart rate (D HR) vs. dermorphin (DER) plasma concentration (mean SEM) following intravenous DER administration. Sampling time is indicated in minutes (m). Predicted data generated using a simple Emax model are shown (solid line). Emax = 27 5 bpm, EC50 = 21.1 8.8 ng/mL.
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Respiratory rate increased by ≥20% over baseline in 4 of 7 horses that were administered DER by the i.v. route from 19 5 to 42 19 breaths per min and returned to baseline within 10 2 min. Intestinal borborygmi were decreased in 6/7 horses (sounds > 1 min apart) between 10 and 40 min post- i.v. administration of DER. Sweating was observed in 4 of 7 horses between 5 and 20 min post- i.v. administration of DER. Profuse sweating with dripping occurred in 2 of these horses, and DER was detected in the sweat collected via a test tube at 7 (0.79 ng/mL) or 13 (0.18 ng/mL) min post- i.v. administration of DER. All 4 horses were dry 1 h after the administration. No changes in rectal temperature were observed. When DER was administered via an i.v. infusion (5 mg at 0.5 lg/kg/min for 15 min), a slow i.v. bolus (5 mg at 8.3 lg/kg over ~1 min), or i.m. (5 mg at 9.2 1.0 lg/kg), changes in HR (Fig. 4b), RR, and sweating were not observed. However, behavioral changes were noted as described above, and intestinal borborygmi were briefly decreased (sounds > 1 min apart) at 12 2 min in 3 of 6 horses administered DER by the i.m. route.
DISCUSSION Combined pharmacokinetic (PK) and pharmacodynamic (PD) studies of DER were not available despite its administration to a number of species (Broccardo et al., 1981; Sander & Giles, 1982; Sandrini et al., 1986; Melchiorri & Negri, 1996). To our knowledge, this is the first combined PK and PD study of DER in a mammal and the first study to evaluate the i.m. route of administration. The presence of DER in postrace horse plasma and urine samples was confirmed by our laboratory with the same methodology utilized in this study (Guan et al., 2013) and prompted this investigation. Following i.v. administration, DER was distributed within a short period (min), and DER concentration was below the LLOQ in plasma within 8 h. Following i.m. administration, DER was rapidly absorbed, and DER concentration was below the LLOQ in plasma within 10 h. The plasma DER concentration measured following i.m. administration could not be fit to the same compartmental model structure in all animals: 4 of 6 animals appeared to fit a 2-compartment elimination model with a distribution phase similar to that observed when DER was intravenously administered, while only a single elimination phase was observed in 2 of 6 animals (models not shown). Thus, noncompartmental modeling was used to estimate the terminal elimination rate constant following i.m. DER administration (Table 1). Complete pharmacokinetic studies of DER using an analytical assay of comparable sensitivity are not available in other species for direct comparison with the results of the present study. Rapid distribution and elimination was observed in rats following i.v. administration with a reported distribution half-life of 1.3 0.4 min and with 95% purity, HPLC-grade water, filtration of the solution prior to administration, and the administration of no more than 5 mL as a bolus, which is 10 000-fold less than the average blood volume reported for horses (10.3 L/100 kg) (Marcilese et al., 1964) render effects due to the vehicle itself an unlikely possibility. The effects of DER appeared to be concentration dependent based on the observations that 1) the effects appeared with increasing dose during the pilot safety study and 2) most of these effects were not observed during a slow i.v. administration of DER (slow push or constant rate infusion) or during the i.m. administration of DER following which the Cmax was 11fold lower than the C0p after a fast i.v. bolus (Table 1). Many of these effects were consistent with those reported for other species administered DER, and similarities were noted between the effects of DER and those of morphine administration to the horse. Behavioral effects of DER in other species are variable and dose dependent, and occur presumably due to the binding of DER to central and/or peripheral l-opioid receptors (Erspamer, 1992; Melchiorri & Negri, 1996). In contrast to humans and monkeys, l-opioid receptor agonists, such as morphine, cause dose-dependent excitation in the horse (Combie et al., 1979). Thus, the excitation observed in horses during the present study was consistent with central activation of the l-opioid receptor by DER. Some of the behavioral changes (head and neck responses) were consistent with those observed as part of © 2014 John Wiley & Sons Ltd
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the well described equine head-shaking syndrome, which is thought to be due to trigeminal neuritis or neuralgia (Newton et al., 2000). Other behavioral changes (tail swishing/smacking, leg lifts toward the belly, head turns back toward the abdomen, with ears turned back) were consistent with colic. In addition, humans administered DER by the i.v. route reported feeling a peripheral tingling sensation, heaviness of limbs, oppression of the chest, and blurred vision (Sandrini et al., 1986). These subjective responses, which may indicate additional effects via binding to peripheral l-opioid receptors, could not be assessed in the horse, and thus, could not be ruled out as possible contributors to the observed behavioral and other physiological responses noted in the present study. Similar to the horses in this study, dogs administered DER (1 or 10 lg/kg) by the i.v. route respond with an acute increase in HR and blood pressure that begins ~30 sec after the bolus, reaches a maximum response at 90–120 sec, and returns to baseline by 5 min (Sander & Giles, 1982). Similarly, humans administered DER as a constant i.v. infusion (5.5 lg/kg/min) for 30 min also respond with statistically significant increases in HR and blood pressure (Degli Uberti et al., 1983), although one subject in a subsequent study had a hypotensive response (Sandrini et al., 1986). When morphine is administered intravenously to the horse, HR and blood pressure increase in a dose-dependent manner for 2 min (50 lg/kg) or 15 min (100 lg/kg or 120 lg/kg) (Muir et al., 1978; Figueiredo et al., 2012). Thus, the increased HR observed in the present study following the administration of DER by the i.v. route to the horse was consistent with DER’s activity as a l-opioid receptor agonist. The exact mechanism(s), however, responsible for the changes in HR observed following DER administration cannot be clearly delineated by the present study, and it is likely that effects of DER on multiple physiological systems contribute to the observed changes in HR. The mean change in HR was plotted against the mean DER plasma concentration and was observed to empirically fit the simple Emax model. This analysis was performed to illustrate the DER plasma concentration observed to elicit an effect on a sensitive physiological variable (HR) and is useful for comparison with plasma concentrations of DER measured in postcompetition samples. It is important to note, however, that there are several limitations to this analysis and that the maximal efficacy and potency of DER was not determined (i.e., the calculated Emax and EC50 are specific to the dose administered and the i.v. route of administration). First, as described above, a clear mechanism for the increased HR resulting from DER administration cannot be identified by this study, and it is likely multifactorial (CNS excitation and/or colic/peripheral effects). Although DER plasma concentration correlates with DHR, this study and analysis do not provide any information regarding the concentration of DER at the l-opioid receptors, the location of the receptors, and does not determine whether the effect on HR is due directly to receptor binding. Second, equilibrium between the undefined biophase and the plasma was not achieved due to the extremely rapid distribution and elimination of DER from © 2014 John Wiley & Sons Ltd
plasma when administered as an i.v. bolus. This means that the DHR observed at each DER plasma concentration may not be representative of the DHR that would be elicited if DER were administered as a constant rate infusion to maintain a designated steady-state plasma concentration. Third, the administration of a single dose does not enable determination of the true maximum effect DER is capable of having on HR (i.e., maximal efficacy was not measured). Larger doses are expected to further increase HR. However, the goal of this study was not to determine the maximal efficacy of DER, and the study of larger doses was not pursed due to safety concerns. Finally, the rapidity of the changes in DER plasma concentration and HR in comparison with the sampling rate do not adequately assess for the possibility of hysteresis. The documented effects of DER administration on RR in this study were more variable than the effects on HR. Morphine administration to the horse increases RR in parallel with the effects on HR and blood pressure (Muir et al., 1978; Figueiredo et al., 2012). As the effects of DER on HR were no longer significant after 5 min, and the first RR recorded in the present study was at ~5 min, the effects on RR may have been missed in some horses. Nostril flaring and agitation prior to 5 min were observed in the video footage of these administrations. The effects of l-opioid receptor agonists on intestinal motility are well described. DER is 40 times more potent than morphine at decreasing intestinal motility in an ex vivo guinea-pig ileum model (Broccardo et al., 1981). In the conscious horse, intestinal motility was easily assessed by auscultation of the abdominal cavity for borborygmi. Similar to the effects of morphine administration (Figueiredo et al., 2012), intestinal borborygmi were decreased (sounds > 1 min apart) following DER administration by the i.v. and i.m. routes in the majority of horses in the study, which was consistent with a direct effect of DER on the intestinal l-opioid receptor. The decrease in borborygmi and the behavioral signs observed suggest acute mild colic may have occurred due to DER administration. To our knowledge, sweating following i.v. administration of DER has not previously been reported in any species. However, flushing and sweating has been reported in some humans following morphine administration (Gutstein & Akil, 2001), and DER increases body temperature of normothermic rats (Broccardo, 1987). Dermorphin administration to the horse did not increase rectal temperature; however, significant sweating was observed in some horses. The presence of DER in paired plasma and urine samples obtained from several horses during routine postrace sample collection was confirmed by our laboratory and 2 others within the USA. All plasma concentrations measured by our laboratory were between 0.005 and 0.020 ng/mL. Although the dose and route of DER administration to the racehorses is not known, the presence of DER in both matrices and at detectable concentrations using the same analytical method as used for this study indicates that the administration (if by the i.v. or i.m. route) was
