Sedative Effects

J. vet. Pharmacol. Therap. 27, 369–372, 2004.

SHORT COMMUNICATION

Sedative effects and serum drug concentrations of oxymorphone and metabolites after subcutaneous administration of a liposome-encapsulated formulation in dogs L. J. SMITH* L. KRUGNER-HIGBY   L. A. TREPANIER à D. E. FLASKA* V. JOERS   & T. D. HEATH § *Department of Surgical Sciences,  Research Animal Resource Center, àDepartment of Medical Sciences, School of Veterinary Medicine and § School of Pharmacy, University of Wisconsin, Madison, WI, USA

(Paper received 19 November 2003; accepted for publication 11 May 2004) Lesley J. Smith, Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive, Madison, WI 53706, USA. E-mail: [email protected] Adequate treatment of chronic pain is increasingly recognized as fundamental to quality companion animal medicine. Companion animals, particularly dogs, can be expected to have an increased lifespan because of modern improvements in veterinary preventative care. With age often come painful chronic conditions such as osteoarthritis and neoplastic disease. The ideal analgesic for treatment of chronic pain would be effective, free from significant side effects, and have a dosage interval and route of administration that is convenient to the caretaker. Encapsulation of drugs into liposomes is one approach to providing a sustained-release drug delivery formulation. Hydrophilic/lipophobic drugs such as morphine have been successfully encapsulated using a multilamellar vesicular system called DepofoamÒ (SkyePharma Inc., San Diego, CA, USA) (Howell, 2001). Oxymorphone is a hydrophobic, lipophilic opioid that is not well suited to encapsulation using multilamellar vesicular technology. We have developed a novel technique for liposomeencapsulation of oxymorphone (Krugner-Higby et al., 2002). This formulation of liposome-encapsulated (LE) oxymorphone releases drug slowly into the systemic circulation from a subcutaneous depot. Oxymorphone is a common, safe, and extremely effective analgesic that is metabolized by the liver with renal and biliary excretion of metabolites (Cone et al., 1983). It is generally well tolerated by a wide patient population. Side effects of oxymorphone in dogs are usually mild at clinically relevant doses, but can include dose-dependent sedation, respiratory depression, panting, bradycardia, nausea, urinary retention, and constipation (Pascoe, 2000a,b; Smith et al., 2001). The objective of the present study was to assess the sedative and physiologic changes associated with subcutaneous administration of LE oxymorphone in healthy dogs and to compare the serum concentration vs. time profiles of LE and standard (STD) oxymorphone. Ó 2004 Blackwell Publishing Ltd

Twenty-five adult dogs, including beagles (n ¼ 14; 12 females, two males) and hounds (n ¼ 11; nine females, two males) aged 1–5 years old (median age 1.5 years) and weighing between 6.9 and 35.7 kg (median weight ¼ 9.46 kg) were used in this study. Normal health status and organ function were confirmed by physical exam and a complete blood count and serum chemistry profile prior to inclusion in the study. None of the animals received any medications in the 2 weeks prior to study. Animals were housed individually (hounds) or in groups of 2–4 (beagles), and were fed a commercial diet (Harlan Teklad 21% Lab Dog Diet (W); Harlan Laboratories, Madison, WI, USA) and water ad libitum throughout the study. The protocol for this study was approved by the University of Wisconsin, School of Veterinary Medicine Animal Care and Use Committee. Standard oxymorphone was purchased from a commercial source (NumorphanÒ Endo Laboratories, Chadds Ford, PA, USA). LE oxymorphone was synthesized using a novel technique previously reported by this laboratory (Smith et al., 2003). Briefly, dehydration–rehydration vesicles containing oxymorphone were prepared by the method of Kirby and Gregoriadis (1984). Oxymorphone in the liposome preparations was quantitated as previously described (Smith et al., 2003). Two doses of STD oxymorphone and LE oxymorphone were investigated. Doses of LE oxymorphone chosen for this study were 0.5 and 1.0 mg/kg, while generally recommended doses of STD oxymorphone are 0.05–0.1 mg/kg for analgesia in dogs (Pascoe, 2000a,b). The proposed equipotent doses of liposomal opioid were designed to be 10 times greater than the commonly recommended doses of the standard formulation, based on previous experience with this formulation (Krugner-Higby et al., 2003; Smith et al., 2003; R. Willis, personal communications), and previous studies using a similar, morphine-based LE preparation in dogs that have employed doses that were 369

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7.5 1 mg/kg LE 0.5mg/kg LE 0.1 mg/kg STD Sedation score

generally ten times greater than those for standard drug preparations of that opioid (Yaksh et al., 1999, 2000). One dose of the liposomal vehicle (unloaded liposomes) was also investigated to confirm the absence of pharmacodynamic properties of the vehicle. Each dog received only one treatment and treatments were administered subcutaneously (s.c.). Treatment groups were as follows: group 1, 1.0 mg/kg LE oxymorphone (n ¼ 6); group 2, 0.5 mg/kg LE oxymorphone (n ¼ 5); group 3, 0.1 mg/kg STD oxymorphone (n ¼ 6); group 4, 0.05 mg/kg STD oxymorphone (n ¼ 6); group 5, 0.7 mL of liposomal vehicle (n ¼ 2). In groups 1 and 2 (LE oxymorphone groups), blood was collected before and 0.5, 1, 2, 4, 8, 12, 16, and 24 h after drug administration, and then daily for 5 days at the same time of day as treatment administration. In groups 3, 4, and 5 (STD oxymorphone and liposomal vehicle groups), blood was collected before and 0.5, 1, 2, 4, 8, 24, and 48 h after drug administration. Blood samples were separated by centrifugation and serum was stored at )70 °C for <1 month until analysis. A sedation score was recorded for each dog prior to drug administration (baseline) and immediately prior to each venous blood collection (Smith et al., 2001). Changes in sedation score, heart rate, respiratory rate, and temperature were analyzed using a Wilcoxon Rank Sum test for nonparametric data with P < 0.05 considered significant. Drug concentrations in serum were quantitated using a commercial ELISA (Neogen Inc., Lexington, KY, USA), which measures both parent drug and the glucuronide metabolites of oxymorphone. The assay yielded an average intra-assay variability of 2.83% and inter-assay variability of 7.61% across the range of expected drug concentrations, i.e. from 0 to 100 ng/mL. The lower limit of quantitation was approximately 1.5 ng/mL. Comparison of the mean serum concentrations achieved at each common time point between groups was performed using ANOVA with post hoc testing using the method of Tukey (SigmaStat, version 2.03, SPSS Science, Chicago, IL, USA). Local skin reactions or obvious infection as a result of injection of the standard or liposomal preparations of drug were not observed in any dog. Injection of the liposomal vehicle had no effect on heart rate, respiratory rate, temperature, or sedation score, and no oxymorphone or metabolite was detected in serum of dogs that received the liposomal vehicle. Figure 1 shows sedation scores in dogs that received LE oxymorphone or STD oxymorphone at the lower and higher doses. Sedation score peaked at 30 min in dogs that received STD oxymorphone, and at 1 h in dogs that received the LE oxymorphone. Sedation score was significantly higher at 1 h in dogs that received 1.0 mg/kg of LE oxymorphone compared with dogs that received either dose of the standard preparation. By 2 h, there was no difference in sedation score between groups. As expected, a mild increase in respiratory rate, manifested as panting, was observed in some dogs, but was not significantly different from baseline for any group. Baseline respiratory rate for all groups was 29.6 ± 2.2 breaths per minute (mean ± SD). At t ¼ 1 h, respiratory rates were: 30.6 ± 18.4 (group 1:

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Fig. 1. Sedation scores in dogs that received either standard (STD) oxymorphone or liposome-encapsulated (LE) oxymorphone. *Significantly higher than the two doses of STD oxymorphone at that time point.

1.0 mg/kg LE oxymorphone); 24.0 ± 2.8 (group 2: 0.5 mg/kg LE oxymorphone); 30.0 ± 16.9 (group 3: 0.1 mg/kg STD oxymorphone); 26.6 ± 2.2 (group 4: 0.05 mg/kg STD oxymorphone); and 26.0 ± 2.8 (group 5: liposomal vehicle). Without access to blood gas analysis, it is not possible to determine whether the observed changes in respiratory rate correlated with significant changes in arterial pH, PaO2 or PaCO2. Mean rectal temperature tended to decrease, although not significantly, from a baseline value (averaged across all groups) of 101.3 ± 1.9–99.7 ± 1.3 °F at t ¼ 1 h in all groups that received some form of oxymorphone. This reduction in temperature may be attributable to the increase in respiratory rate, or panting, in some dogs. Figure 2 shows the changes in heart rate that occurred after administration of the LE oxymorphone or STD oxymorphone. A significant decrease in heart rate was observed from baseline to 2 h after all doses of LE or STD oxymorphone were administered. The lowest heart rate observed (64 bpm) was at t ¼ 1 h in one dog that received 1.0 mg/kg of LE oxymorphone. In all groups that received any form of oxymorphone, actual heart rates were never less than what would be considered to be within a clinically acceptable range for healthy dogs. Figure 3 shows serum concentrations of oxymorphone and its glucuronide in all four groups of dogs. Serum concentrations were significantly higher in the dogs that received 1.0 mg/kg of LE oxymorphone at all time points measured. By 24 h, concentrations of drug in dogs that received either dose of STD oxymorphone or the lower dose of LE oxymorphone fell below the limit of quantitation (1.5 ng/mL). The higher dose of LE oxymorphone therefore resulted in more persistent serum concentrations of both parent drug and metabolites than either the lower dose of LE drug or either dose of the standard preparation. Currently available opioid analgesics that can be prescribed for Ôat homeÕ use in the US and that provide an extended duration of effect include the fentanyl patch, and oral formulations of butorphanol, morphine, oxycodone, oxycontin, and codeine. All of these opioid formulations present a significant risk of drug diversion by human caretakers (Purucker & Swann, 2000; Ó 2004 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 27, 369–372

Liposome-encapsulated oxymorphone in dogs 371 1 mg/kg LE 0.5 mg/kg LE 0.1mg/kg STD 0.05mg/kg STD

Fig. 2. Changes in heart rate in dogs that received either standard (STD) oxymorphone or liposome-encapsulated (LE) oxymorphone. *Significantly different than baseline across groups; **significantly different from other groups at that time point.

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Fig. 3. Serum oxymorphone and metabolite concentrations in all groups of dogs from baseline to 5 days postadministration of oxymorphone. *Significantly different than the lower doses of STD and LE oxymorphone.

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Passik, 2001; Wasserman, 2001) or accidental access to children (Hardwick et al., 1997). In addition, bioavailability and efficacy of these opioid formulations are variable in humans (Gourlay et al., 1997) and dogs (Kyles et al., 1996; Dohoo, 1997; Egger et al., 1998). There is a need for a long acting, physiologically safe, and effective analgesic for dogs that can be administered without the risk of illicit access by caretakers. The purpose of the current study was to assess the sedative effects and the serum concentration vs. time behavior of a single subcutaneous injection of LE oxymorphone, and to compare these results to those measured after subcutaneous injection of an equipotent dose of the standard, aqueous formulation of oxymorphone in dogs. Our data indicate that administration of 1 mg/kg of LE oxymorphone to healthy dogs resulted in moderate, transient bradycardia and significant, but clinically acceptable, sedation at 1 h after subcutaneous administration. The significant sedation observed at 1 h after administration of the higher dose of LE oxymorphone correlates with the higher serum drug concentrations seen with this dosing regimen. The lack of a significant difference in sedation at the 30-min assessment point, despite serum oxymorphone concentrations that were comparable with the 1 h time point, may have been due to an effect lag as oxymorphone diffused to the CNS. Sedation scores were similar in all dogs at 2 h after administration of either 1.0 mg/kg LE oxymorphone, or one-tenth that dose (0.1 mg/kg) of STD oxymorphone. Of note is the lack of significant differences in Ó 2004 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 27, 369–372

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sedation at later time points, despite a consistently higher oxymorphone concentration throughout the sampling period. This may reflect the presence of oxymorphone glucuronide, which may have less sedative effects than the parent drug. Using an ELISA assay that measures both oxymorphone and its glucuronide metabolite, we observed serum concentrations after administration of 0.1 mg/kg STD oxymorphone of 19.1 ± 11.2 and 11.1 ± 3.6 ng/mL (mean ± SD) at 2 and 4 h, respectively (see Fig. 3). There are no published studies that have correlated serum concentrations of oxymorphone with clinical analgesia in dogs. Current dosing recommendations for STD oxymorphone in dogs, however, are 0.05–0.1 mg/kg administered every 3–4 h (Pascoe, 2000a,b). This would imply that serum concentrations of oxymorphone are maintained within an apparent therapeutic range for up to 4 h after administration. From these clinical practices, along with our data in the current study, we would infer that serum concentrations of total oxymorphone (parent drug and metabolites) less than 10 ng/mL are below the therapeutic range. After administration of what we estimated was an equipotent dose of LE oxymorphone (1.0 mg/kg), we observed serum concentrations of drug >10 ng/mL for over 2 days after a single injection. It is important to note that because our assay measures both parent drug and glucuronide metabolites, we cannot state that measured total drug concentrations correspond directly to pharmacologic activity. Without concurrent algesiometry, or a more specific assay, such as HPLC, we cannot equate the

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reported serum concentrations with clinical effectiveness. It will be important to follow-up on these studies with analgesic testing using a variety of experimental stimuli in dogs treated with liposomal oxymorphone. We are also developing a more specific HPLC assay to clarify the relationship between serum drug concentrations and pharmacodynamic effects. The results of this study indicate that the subcutaneous administration of 1.0 mg/kg LE oxymorphone is not associated with excessive sedation in healthy dogs, and results in total serum drug concentrations >10 ng/mL for at least 2 days after a single dose. These concentrations are comparable with those sustained for only 4 h after a single dose of STD oxymorphone. More specific pharmacokinetic and pharmacodynamic studies are warranted to test this liposomal formulation of oxymorphone for analgesic efficacy, and for safety in geriatric animals or those with systemic disease. The results of this preliminary study have significant potential implications for pain management in companion animal and human medicine. Liposome-encapsulation of oxymorphone provides a formulation of drug that can potentially be administered at delayed intervals and that may induce fewer side effects than standard formulations of opioids due to the avoidance of bolus absorption to therapeutic concentrations. Clearly, more studies in companion animals, and, ultimately, humans are warranted to define the pharmacokinetic and analgesic properties of LE oxymorphone.

ACKNOWLEDGMENTS Funded by the Companion Animal Foundation, University of Wisconsin, School of Veterinary Medicine, and the American College of Laboratory Animal Medicine. The authors wish to thank Dr Barb Gilligan and Dr Jim Southard of the Transplant Research Laboratory, University of Wisconsin Medical School and Dr Mark Markel and the Comparative Orthopedic Research Laboratory, University of Wisconsin School of Veterinary Medicine for their generous provision of dogs for this study, and Claudia Hirsch and the animal care staff at Charmany Instructional Facility for their care of animals.

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Gourlay, G.K., Cherry, D.A., Onley, M.M., Tordoff, S.G., Conn, D.A., Hood, G.M. & Plummer, J.L. (1997) Pharmacokinetics and pharmacodynamics of twenty-four-hourly Kapanol compared to twelve-hourly MS Contin in the treatment of severe cancer pain. Pain, 69, 295–302. Hardwick, W.E., King, W.D. & Palmisano, P.A. (1997) Respiratory depression in a child unintentionally exposed to transdermal fentanyl patch. Southern Medical Journal, 90, 962–964. Howell, S.B. (2001) Clinical applications of a novel sustained-release injectable drug delivery system: DepofoamTM technology. Cancer Journal, 7, 219–227. Kirby, C.J. & Gregoriadis, G. (1984) A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions. In Liposome Technology, Vol. I. Ed. Gregoriadis, G. pp. 19–27. CRC Press, Boca Raton, FL. Krugner-Higby, L., Smith, L.J. & Heath, T. (2002) Liposome-encapsulated oxymorphone hydrochloride for long-term analgesia. United States Patent Application 20030157162; Krugner-Higby, L., Heath, T.D. & Smith, L.J.; Wisconsin Alumnae Research Foundation, Madison, WI 53706, USA. Krugner-Higby, L., Smith, L.J., Clark, M., Heath, T.D., Dahly, E., Schiffman, B., Hubbard-VanStelle, S., Ney, D. & Wendland, A. (2003) Liposome-encapsulated oxymorphone hydrochloride provides prolonged relief of post-surgical visceral pain in rats. Comparative Medicine, 53, 270–280. Kyles, A.E., Papich, M. & Hardie, E.M. (1996) Disposition of transdermally administered fentanyl in dogs. American Journal of Veterinary Research, 57, 715–719. Purucker, M. & Swann, W. (2000) Potential for duragesic patch abuse. Annals of Emergency Medicine, 35, 314. Pascoe, P.J. (2000a) Opioid analgesics. In Pain Management: Veterinary Clinics of North America, Small Animal Practice Ed. Matthews, K.A. pp. 757–772. WB Saunders Co., Philadelphia, PA. Pascoe, P.J. (2000b) Perioperative pain management. In Management of Pain: Veterinary Clinics of North America, Small Animal Practice Ed. Matthews, K.A. pp. 917–933. WB Saunders Co., Philadelphia, PA. Passik, S.D. (2001) Responding rationally to recent report of abuse/ diversion of oxycontin. Journal of Pain and Symptom Management, 21, 359. Smith, L.J., Yu, J.K.-A., Bjorling, D.E. & Waller, K. (2001) Effects of hydromorphone or oxymorphone, with or without acepromazine, on preanesthetic sedation, physiologic values, and histamine release in dogs. Journal of the American Veterinary Medical Association, 218, 1101– 1105. Smith, L.J., Krugner-Higby, L., Clark, M., Wendland, A. & Heath, T.D. (2003) Liposome-encapsulated oxymorphone provides prolonged analgesia in an animal model of neuropathic pain. Comparative Medicine, 53, 280–288. Wasserman, S. (2001) States respond to growing abuse of painkiller. State Legislatures, 27, 33–34. Yaksh, T.L., Provencher, J.C., Rathbun, M.L. & Kohn, F.R. (1999) Pharmacokinetics and efficacy of epidurally delivered sustained-release encapsulated morphine in dogs. Anesthesiology, 90, 1402–1412. Yaksh, T.L., Provencher, J.C., Rathbun, M.L., Myers, R.R., Powell, H., Richter, P. & Kohn, F.R. (2000) Safety assessment of encapsulated morphine delivered epidurally in a sustained-release multivesicular liposome preparation in dogs. Drug Delivery, 7, 27–36.

Ó 2004 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 27, 369–372