Experimental Pharmacodynamics and Analgesic Efficacy of Liposome-Encapsulated Hydromorphone in Dogs
The purpose of this study was to determine the experimental side effects of liposome-encapsulated hydromorphone (LE-Hydro) in beagles and to evaluate LE-Hydro analgesia in dogs undergoing ovariohysterectomies (OVH). Beagles were injected subcutaneously with 1–3 mg/kg LE-Hydro or 0.1 mg/kg hydromorphone. Dogs were evaluated for sedation, temperature, respiratory rate, and heart rate. OVH dogs were injected with 2 mg/kg LE-Hydro subcutaneously or 0.2 mg/kg morphine and 0.05 mg/kg acepromazine intramuscularly. Side effects of LE-Hydro were within clinically acceptable limits. The analgesic efficacy was superior in dogs administered LE-Hydro at 12 hr postsurgically. LE-Hydro provided adequate, durable analgesia in dogs undergoing OVH.
Introduction
Ovariohysterectomy (OVH) is one of the most common operative procedures performed on dogs and cats. Along with castration, OVH is the most common surgical procedure performed in humane societies and animal shelters.1 Shelters receive many different breeds of dogs that must be sterilized prior to adoption. The conditions of the animal shelter environment require that the analgesics used for operative and postoperative use be inexpensive, simple to administer, cause minimal side effects, and be effective throughout the entire postoperative period. Single agent regimens are simpler to administer, but balanced analgesia, where multiple analgesics with different mechanisms of action are combined, can potentially provide more effective analgesia.2 These two opposing issues must be weighed carefully in the shelter environment.
OVH is also one of the most common surgical procedures used to test analgesics for the control of pain during and after surgery. Most published studies compare the effects of commercially manufactured drugs already used in veterinary medicine. Recent studies have compared: morphine and carprofen (alone or in combination); ketoprofen and carprofen; ketoprofen, oxymorphone, and butorphanol; butorphanol and meloxicam; epidurally administered levogyral ketamine and morphine (alone or in combination); and carprofen and pethidine (alone or in combination).3–8 OVH is performed frequently in both private clinics and academic veterinary hospitals to provide sufficient numbers of cases for such comparisons. Further, the surgery can be performed consistently enough to make such comparisons valid.
The first liposome-encapsulated commercial drug formulations were made using the cancer chemotherapy agents doxorubicin and cytarabine, as well as the antifungal drug amphotericin B.9–11 The chief advantages to these formulations were their decreased toxicity compared with the standard formulations of these drugs. Liposome-encapsulated formulations of opioid drugs such as oxymorphone and hydromorphone have been shown to provide longer release kinetics, fewer undesirable side effects, and superior efficacy compared with either the standard formulations of the same opioid drug or the partial μ agonist opioid buprenorphine, which is widely used in veterinary practice because of its prolonged duration.12–14 Liposomal analgesics used as an extended-release depot may provide analgesia without indwelling intravenous (IV) or epidural catheters, a considerable advantage in veterinary medicine. Liposome-encapsulated morphine sulfate was tested on dogs using a skin-flick analgesiometry model during the preclinical development of the formulation, but there has not been a published study using it on dogs in clinical veterinary practice.15,16 Extended-release pain medication would also have the advantage that no opioid medication would be placed in the client's hands, where it may be diverted for abuse or accidentally ingested by children. Liposome-encapsulated hydromorphone (LE-Hydro), made with dipalmitoylphosphatidylcholine and cholesterol in the membrane, has been shown to provide an early peak of drug release, followed by sustained serum concentrations of at least 4 ng/mL (depending on the dose administered) for up to 96 hr in dogs after a single subcutaneous (SC) injection.12
This study compared the side effects profile of LE-Hydro with that of the standard formulation of hydromorphone in healthy beagles. After the side effects profile of LE-Hydro had been determined, the analgesic efficacy of a single SC injection of LE-Hydro (2 mg/kg) was evaluated as the sole analgesic in dogs undergoing OVH in an animal shelter prior to adoption. LE-Hydro was compared with the standard analgesic regimen in the facility, a combination of a single intramuscular (IM) injection of morphine sulfate (0.2 mg/kg) and acepromazine maleate (0.05 mg/kg). In addition to testing this novel, extended-release formulation of hydromorphone in dogs undergoing OVH, the drug was evaluated for use in a county animal shelter, the type of environment where extended-release pain medication may have considerable advantages for animals and personnel. The purpose of this study was to test the hypotheses that LE-Hydro will show a dose-response, have an acceptable side effects profile, and that LE-Hydro would be an effective analgesic for OVH in shelter dogs.
Materials and Methods
Preparation of Liposome-Encapsulated Hydromorphone Using Modified Dehydration-Rehydration Vesicles
Liposomes containing hydromorphone HCla were prepared using previously published methods.12 Briefly, dipalmitoylphosphatidylcholineb and cholesterolb were dried from a chloroform solution in a 20 mm screw cap tube. The lipid was dissolved in 1 mL of sterile tert-butanola by heating to 55°C in a water bath, and lyophilized for 24 hr. The dried lipid was swollen in 1 mL of 40 mg/mL hydromorphone HCla, frozen using dry ice and isopropanol for 2 min then stored at -20°C until time of use. When needed, the liposomes were thawed at room temperature, separated from the unencapsulated drug, and analyzed for hydromorphone content as previously described.12 The suspension was stored in a dark cabinet at 4°C for no more than 7 days prior to use. Immediately prior to injection into an animal, the preparation was gently agitated then slowly drawn up into a syringe using a 22 gauge needle.
Side Effects Profile of LE-Hydro
The University of Wisconsin School of Veterinary Medicine Research Animal Care and Use Committee approved all studies. The animals used for these studies have been described in detail.12 Healthy, purpose-bred, male neutered Beagle dogsc were used (n=6–7/group). Normal health status was confirmed prior to entry into the study. A vascular access port was surgically placed between the dogs' shoulder blades and the dogs were neutered. The dogs were allowed to recover for 30 days before study initiation. Dosing protocols were similar to those used for pharmacokinetic studies.12 Briefly, SC drugs were administered at the loose skin caudal to the scapulae at least 5 cm distal to the permanent vascular access port. The following groups were studied: 0.1 mg/kg hydromorphone HCl SC(n=6); 1.0 mg/kg LE-Hydro SC (LE-Hydro-1, n=6); 2.0 mg/kg LE-Hydro SC (LE-Hydro-2, n=6); and 3.0 mg/kg LE-Hydro SC (LE-Hydro-3, n=7). The same six dogs were randomized to receive hydromorphone, 1.0 mg/kg LE-Hydro, and 2.0 mg/kg LE-Hydro. For the 3 mg/kg LE-Hydro group, two dogs were used as pilot animals to assess the sedative effects of the higher dose. That data were pooled with that of five additional dogs administered 3.0 mg/kg LE-Hydro. The five dogs in the 3 mg/kg group did not receive hydromorphone or any other dose of LE-Hydro. This protocol was selected because the dogs used for the earlier studies had attained their maximum approved research use and had been adopted out of the facility. The dogs that were administered multiple treatments were allowed a 2–4 wk washout between each treatment.
Dogs were evaluated for sedation using a standard numerical behavioral scoring tool and a visual analog scale (VAS). The behavioral scoring tool had a scale of -11 to 10. Scores <0 were indicative of increasing sedation and scores >0 were indicative of increasing excitation. The VAS was a scale of 0–10. Increasing scores were indicative of increasing sedation. Measurements of rectal temperature, respiratory rate, and heart rate were also taken. Occurrences of nausea, defecation, dysphoria, and panting were also recorded (Figure 1). Data were collected before and at serial time points after drug administration (i.e., at baseline then 5min, 10 min, 15 min, 20 min, 30 min, 45min, 60 min, 90min, 120 min, 4 hr, 6 hr, 8 hr, 12 hr, 24 hr, 48 hr, 72 hr, 96 hr, 120 hr, and 144 hr). Data were collected by trained observers (B.S., L.W.) who were blind to treatment condition in this phase of the study.
Citation: Journal of the American Animal Hospital Association 47, 3; 10.5326/JAAHA-MS-5599
Safety and Efficacy of LE-Hydro for OVH
Dogs used for this portion of the study were community source animals surrendered to a municipal animal shelter. The dogs were evaluated for general health by physical examination and for temperament to exclude aggressive animals prior to surgery. All dogs were negative for antigens to Dirofilaria immitis. Dogs (n=10/group) were randomly assigned to receive either 2.0 mg/kg LE-Hydro administered SC or the standard of care for the shelter facility (i.e., 0.2 mg/kg morphine sulfate and 0.05 mg/kg acepromazine maleate administered IM). All test drugs were administered 20–30 min prior to anesthetic induction for purposes of OVH. At extubation (time=0) and again at 30 min, 60 min, 2 hr, 6 hr, 12 hr, and 36 hr after extubation, the dogs' heart rates, respiratory rates, and rectal temperatures were recorded. At the same time points, a numerical rating pain score was recorded by trained observers (B.S., L.W., A.S.) and incisional pain was assessed using an electronic von Frey meterd. The pressure point (i.e., the blunt end of a pipette tip) of the mechanical von Frey meter was pressed 1 cm on either side of the incision at the cranial, middle, and most caudal points until a purposeful reaction from the dog was elicited (i.e., head turn, flinch, vocalization, or moving away). A maximal threshold of 1000 g was used as a cutoff point.
The pain scoring sheet used for this study (shown in Figure 2) was a modification of the University of Melbourne Pain Scoring form, which has been validated by the authors' laboratory and others.17,18 Observers were instructed in applications of the sedation and pain scoring systems and all observers had participated in multiple pharmacokinetic and pharmacodynamic studies prior to using these tools on the OVH dogs included in the present study. Sedation and pain scores were performed independently by two different observers at each time point and averaged.
Citation: Journal of the American Animal Hospital Association 47, 3; 10.5326/JAAHA-MS-5599
A number of contingencies were provided for in this study. First, any dog determined by the Dane county Humane Society (DCHS) veterinary staff or the researchers to have excessive sedation, bradycardia, respiratory depression, or dysphoria after administration of LE-Hydro were given 0.2 mg/kg butorphanol tartrate SC. Second, in both treatment groups, rescue analgesia for unacceptable pain behavior (0.5–1.0 mg/kg morphine sulfate IM) was administered by either the DCHS veterinary staff or by the researchers at their discretion.
This study was an open label trial. This was the first trial of LE-Hydro in random source dogs in a nonlaboratory setting and the researchers and the personnel at the shelter needed to know which treatments the dogs received to respond adequately to potential problems. The study authors were required by the shelter to use the shelter's standard analgesic regimen. No changes were allowed in the standard morphine and acepromazine analgesic combination administered to the shelter dogs (that were not administered the LE-Hydro).
Statistical Analysis
Side effects data from healthy beagles were analyzed without transformation. Body temperature, heart rate, respiratory rate, sedation scores and VAS were analyzed for differences within groups to compare values over time to baseline values using the Wilcoxon signed rank test. This test was selected as it provided a robust, conservative analysis and because the data were not normally distributed. The Mann-Whitney U test was used for comparisons between groups (1 mg/kg LE-Hydro versus 2 mg/kg LE-Hydro; 1 mg/kg LE-Hydro versus 3 mg/kg LE-Hydro; and 2 mg/kg LE-Hydro versus 3 mg/kg LE-Hydro), and a Bonferroni adjustment for multiple comparisons was applied to the dose-response data (critical P value was ≤0.017). The frequency of occurrence of side effects such a nausea, defecation, and dysphoria were analyzed using a binomial test. Data from the DCHS study were also analyzed using the Wilcoxon signed rank test for within groups comparisons and the Mann-Whitney U test for between groups comparisons because the data were not normally distributed. All statistical analyses were run on commercial statistical softwaree. Significance was inferred at P≤0.05 except where noted above for the Bonferroni correction.
Results
Studies in beagles indicated that the side effects of LE-Hydro were consistent with those seen for all pure μ agonist opioid drugs. Analysis of numerical sedation and VAS scores showed typical dose-dependent increases in sedative effects (Table 1). Within group analysis of behavioral and physiologic values from dogs administered hydromorphone showed expected changes from baseline values for that group. The LE-Hydro-1 group had less sedation and fewer changes in respiratory rate, heart rate, and body temperature compared with baseline values than for dogs administered the higher doses of LE-Hydro. Intermediate values were obtained in the LE-Hydro-2 group. The most persistent changes were observed in the LE-Hydro-3 group compared with baseline (Table 1, Table 2). Analysis within and between groups indicated that the most profound and persistent changes occurred in beagles administered the 3 mg/kg dose of LE-Hydro. Changes in measures of sedation and physiologic variables were lowest in dogs administered 1 mg/kg LE-Hydro, intermediate in 2 mg/kg LE-Hydro group, and highest in the 3 mg/kg LE-Hydro group (Tables 1, 2).
Indicates statistical significance using the Wilcoxon signed rank test when compared within treatment groups indicating that the values were different from baseline for that group
Indicates significance for comparisons between groups using the Mann-Whitney U test (the only significant comparisons were 1 mg/kg LE-Hydro versus 3 mg/kg LE-Hydro).
LE-Hydro, liposome-encapsulated hydromorphone
Indicates statistical significance using the Wilcoxon signed rank test when compared within treatment groups indicating that the values were different from baseline for that group.
Indicates significance for comparisons between groups using the Mann-Whitney U test (the only significant comparisons were 1 mg/kg LE-Hydro versus 3 mg/kg LE-Hydro).
LE-Hydro, liposome-encapsulated hydromorphone
Based on the pharmacokinetics and the side effects profile of LE-Hydro observed in the laboratory studies in the beagles, the 2 mg/kg dosage was chosen for use in the open label clinical trial of LE-Hydro in dogs undergoing OVH at a municipal animal shelter.12 None of the dogs observed by the DCHS staff or the researchers required rescue pain medication or butorphanol for either excessive sedation or dysphoria. The occurrence of nausea and defecation were similar in both the dogs administered the morphine and acepromazine combination and the LE-Hydro dogs (data not shown). The frequency of these side effects in the LE-Hydro dogs was consistent with what was observed in beagles administered the same 2 mg/kg dose in the earlier study (Table 3). Dysphoria was observed in three pinscher breed dogs (one Doberman pinscher and two miniature pinschers). All three pinscher breed dogs used in the study were randomized to the LE-Hydro group. No pinscher breed dogs were randomized to the group administered the morphine and acepromazine combination.
Indicates statistical significance using a binomial Test when LE-Hydro-1 was compared with the other three treatment groups (i.e., hydromorphone, LE-Hydro-2, and LE-Hydro-3).
Indicates statistical significance using a binomial Test when LE-Hydro-3 was compared with the 1 and 2 mg/kg LE-Hydro groups.
LE-Hydro, liposome-encapsulated hydromorphone
Analysis of the data indicated that pain scores were significantly higher for the LE-Hydro dogs at 30 min, 45 min, and 60 min, but were lower 12 hr after surgery compared with baseline values (Table 4). The von Frey meter scores for LE-Hydro dogs were increased over baseline for the first 30 min after surgery, suggesting pain thresholds were increased compared with baseline. Von Frey scores after surgery were close to baseline in the group administered the morphine and acepromazine combination and remained so until 12 hr postsurgically, at which time von Frey scores were significantly decreased. This finding suggested a decrease in pain thresholds. Both groups appeared to develop mild hypersensitivity at the skin wound by 24 hr, as evidenced by decreasing von Frey values, but there was no significant difference between the two groups beyond 12 hr after surgery (Table 4).
Indicates statistical significance from baseline values for that group using the Wilcoxon signed rank test when compared within treatment groups.
Indicates significance for comparisons between LE-Hydro versus morphine and acepromazine.
LE-Hydro, liposome-encapsulated hydromorphone
Body temperature was not significantly different between the two groups of dogs. Respiratory rates were significantly higher in dogs administered LE-Hydro at 30 min and at 12 hr after surgery. The heart rates of dogs administered LE-Hydro were only significantly increased at 15 min after surgery (Table 4).
Discussion
LE-Hydro provides serum concentrations in dogs that are within the therapeutic range for people for 4 days after a single subcutaneous injection.12 This concentration is assumed to be therapeutic for dogs as well based on the pharmacokinetics of standard hydromorphone in dogs and the dosages of this drug used in clinical veterinary practice.19–21 Further study of hydromorphone is warranted in veterinary clinical patients before therapeutic concentrations can be definitively determined for dogs. The results of the current study indicate that LE-Hydro achieves therapeutic concentrations for treatment of acute, postsurgical OVH-associated pain. None of the dogs required supplemental pain medication. Other types of extended-release formulations may not achieve this goal. Hydromorphone incorporated into ethylene vinyl acetate copolymer disks provides low, but sustained, plasma drug concentrations for up to 4 wk after SC injection in rabbits.22 This type of extended-release preparation does not provide high enough initial concentrations to be sufficient for treating postoperative pain and would be more appropriate for treating chronic pain.
There were no adverse reactions in any of the dogs that were administered LE-Hydro requiring staff intervention. In the OVH portion of this study, based on pain score data, the LE-Hydro dogs had significantly higher pain scores 30–60 min after surgery. At these time points, however, the dogs had relatively high von Frey thresholds and lower heart rates than the dogs administered the standard morphine and acepromazine combination at the same time points, indicating that the dogs may not have been truly uncomfortable (Table 4). There were also three pinscher breed dogs in the LE-Hydro group. These dogs were anxious and agitated in their cage, which may have skewed the pain scores for the LE-Hydro group to be higher than for the control group of dogs. The type of surgical procedure used to evaluate the two analgesic regimens and the ethographic scoring system used to evaluate the dogs must also be taken into account. It is possible that subtle differences in pain between the two groups could not be detected using the pain scoring system used in this study or given the nature of the postoperative pain.
OVH can be expected to cause mild to moderate pain. Thus, subtle differences between analgesic regimens may not be as apparent as the differences that might be found using a more invasive surgical procedure that causes moderate to severe pain. Studies comparing groups of dogs undergoing pelvic or femoral fracture repair found a difference between groups administered oral carprofen alone or epidural mepivacaine (either alone or in combination with carprofen).23 The disadvantages of this latter type of study is that the dogs would be more likely to have other complications from the inciting trauma, making the animals more difficult to evaluate. Further, orthopedic procedures are not often performed in municipal animal shelters.
Ethographic scoring tools also may not differentiate degrees of pain very effectively between pain control regimens that are reasonably adequate. Humans have been reported to be able to differentiate up to 21 different degrees of pain. Using the pain scoring tools currently available to veterinary investigators and clinicians, it is only possible to differentiate up to three or four degrees of pain, a considerable loss of sensitivity.24 Questions may arise as to how well pain can be quantitated in animals. The modified University of Melbourne Pain Scoring form has been used in a number of studies in dogs.17,18 In a study evaluating lidocaine infusion for analgesia after enucleation, 100% of the control dogs received rescue pain medication based on scores determined using this form. Only 50% dogs given either morphine injections or the lidocaine infusion received rescue medication.17 In the current study, the largest differences between LE-Hydro and the morphine and acepromazine combination with respect to both pain scores and von Frey values were recorded 12 hr after surgery. At that time point, the morphine would be expected to be at subtherapeutic concentrations, but postoperative pain would still be severe enough to require medication (Table 4). Current ethographic and VAS systems differentiate between adequate and poor analgesic regimens, but do not differentiate well between analgesic regimens that are at least adequate.
VAS and ethographic scoring systems with verbal descriptions have been used to rate sedation and pain in animals and humans.20,25,26 These tools have also been used to assess lameness in domestic animals including sheep, horses, and dogs.27–29 The VAS has been found to be a more sensitive measure for pain and lameness in animals than ethographic scoring. In the current pharmacodynamics study using beagles, the VAS identified one additional time point as being significantly different for both within and between groups analyses of sedation, not very different than the results using ethographic scoring (Tables 1, 4). Although they are easy tests to perform and are widely used, both types of behavioral evaluations are not as sensitive as more objective measures such as pain threshold testing or force plate for lameness.25,29
The pain relief provided by the morphine and acepromazine combination was better and more durable than expected (based on the pain scoring systems used in this study). Morphine sulfate alone, injected IM at the dose used in this study, would be expected to be effective for <4 hr in dogs.30 The change in pain scores and von Frey (Table 4) measurements between dogs that received LE-Hydro and the morphine and acepromazine combination that occurred between 8 hr and 12 hr postoperatively indicated that the morphine and acepromazine combination was effective for at least 8 hr after surgery. This is twice the effective period of analgesia predicted from the pharmacokinetics of the morphine alone. This increased duration of analgesia was probably due to the coadministration of acepromazine. Acepromazine is a phenothiazine derivative tranquilizer that decreases arterial blood pressure and cardiovascular function and is a potent peripheral vasodilator that decreases hepatic blood flow.31 Despite the wide use of acepromazine in dogs, published pharmacokinetics for this species are not available. Pharmacokinetic analysis of acepromazine in horses indicate that the half-life of this drug is 1.6–3 hr, so full clearance from the circulation may take up to 15 hr.32,33 The liver metabolizes opioid drugs. Thus, any drug that decreases hepatic blood flow will increase the effective half-life of morphine. This effect may have been exacerbated by general anesthesia with isoflurane.
Conclusion
The ideal analgesic for use in a shelter environment needs to be effective as a sole agent, effective for the entire operative and postoperative periods, and be safe enough to require only minimal monitoring by the veterinary staff postprocedurally. LE-Hydro fulfilled each of these criteria. This open-label study is the first to evaluate the efficacy of LE-Hydro for postsurgical pain in dogs. A larger, more comprehensive clinical trial is currently underway in dogs undergoing limb amputation (L.S., unpublished data).
Sedation scoring sheet used for assessing dogs administered either hydromorphone or liposome-encapsulated hydromorphone (LE-Hydro).
Ethogram used for dogs undergoing ovariohysterectomy.
Contributor Notes
