Multiple-dose pharmacokinetics and opioid effects of a novel analgesic with a deterrent to human opioid abuse (methadone-fluconazole-naltrexone) after oral administration in dogs

Butch KuKanich Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502.

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 DVM, PhD
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Kate KuKanich Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502.

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Charles W. Locuson Vanderbilt University Center for Neuroscience Drug Discovery, Cool Springs Life Sciences Center, Nashville, TN 37240.

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David C. Rankin Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502.

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Alyson H. Fitzgerald Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502.

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Peter Cho Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502.

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Marissa S. Komp Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502.

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Abstract

OBJECTIVE

To assess the pharmacokinetics and opioid effects of methadone after administration of multiple doses by means of 2 dosing regimens of methadone-fluconazole-naltrexone.

ANIMALS

12 healthy Beagles.

PROCEDURES

Dogs were randomly allocated (6 dogs/group) to receive 1 of 2 oral dosing regimens of methadone-fluconazole-naltrexone. Treatment 1 doses were administered at 0 (methadone-to-fluconazole-to-naltrexone ratio of 1:5:0.25 mg/kg), 14 (1:5:0.25), 24 (0.5:2.5:0.125), and 38 (0.5:2.5:0.125) hours. Treatment 2 doses were administered at 0 (1:5:0.25), 4 (0.5:2.5:0.125), 10 (0.5:2.5:0.125), and 24 (0.5:2.5:0.125) hours. Blood samples, rectal temperatures, and von Frey antinociceptive measurements were obtained at designated times.

RESULTS

Compared with baseline, temperatures significantly decreased for treatment 1 group dogs at 2 to ≥ 4 hours and from 16 to ≥ 50 hours (12 hours after last dose) and for treatment 2 group dogs at 2 to ≥ 36 hours (12 hours after last dose), when trough methadone concentrations were ≥ 21.3 ng/mL. Antinociception occurred after the first dose but was not maintained throughout the study. Lesions were noted in some dogs at the application site of the von Frey device. Naltrexone and β-naltrexol were sporadically detected in plasma, and naltrexone glucuronide was consistently detected.

CONCLUSIONS AND CLINICAL RELEVANCE

Opioid effects were noted after oral administration of the first dose, and data suggested that administering a second dose 6 hours later and every 12 hours thereafter was necessary to maintain opioid effects. Antinociception may have been lost because dogs became averse or hyperalgesic to the von Frey device, such that the antinociception model used here may not be robust for repeated measurements in dogs.

Abstract

OBJECTIVE

To assess the pharmacokinetics and opioid effects of methadone after administration of multiple doses by means of 2 dosing regimens of methadone-fluconazole-naltrexone.

ANIMALS

12 healthy Beagles.

PROCEDURES

Dogs were randomly allocated (6 dogs/group) to receive 1 of 2 oral dosing regimens of methadone-fluconazole-naltrexone. Treatment 1 doses were administered at 0 (methadone-to-fluconazole-to-naltrexone ratio of 1:5:0.25 mg/kg), 14 (1:5:0.25), 24 (0.5:2.5:0.125), and 38 (0.5:2.5:0.125) hours. Treatment 2 doses were administered at 0 (1:5:0.25), 4 (0.5:2.5:0.125), 10 (0.5:2.5:0.125), and 24 (0.5:2.5:0.125) hours. Blood samples, rectal temperatures, and von Frey antinociceptive measurements were obtained at designated times.

RESULTS

Compared with baseline, temperatures significantly decreased for treatment 1 group dogs at 2 to ≥ 4 hours and from 16 to ≥ 50 hours (12 hours after last dose) and for treatment 2 group dogs at 2 to ≥ 36 hours (12 hours after last dose), when trough methadone concentrations were ≥ 21.3 ng/mL. Antinociception occurred after the first dose but was not maintained throughout the study. Lesions were noted in some dogs at the application site of the von Frey device. Naltrexone and β-naltrexol were sporadically detected in plasma, and naltrexone glucuronide was consistently detected.

CONCLUSIONS AND CLINICAL RELEVANCE

Opioid effects were noted after oral administration of the first dose, and data suggested that administering a second dose 6 hours later and every 12 hours thereafter was necessary to maintain opioid effects. Antinociception may have been lost because dogs became averse or hyperalgesic to the von Frey device, such that the antinociception model used here may not be robust for repeated measurements in dogs.

Introduction

μ-Opioid receptor agonists are safe and highly effective analgesics for amelioration of signs of acute pain in dogs. However, their use is limited because of their poor oral bioavailability, short half-life, and short duration of effect.1 Opioids are also thought to affect the hypothalamus and reduce body temperature in a dose− and concentration-dependent manner that parallels their antinociceptive effects.2,3 As such, measuring decreases in body (rectal) temperature can be used as a relatively noninvasive marker of central opioid effects.

Methadone is an opioid approved in some countries as a safe and effective analgesic for parenteral administration in dogs. The pharmacokinetics of methadone in dogs have been well described, and methadone's half-life has been confirmed to be short and its oral bioavailability low, similar to other μ-opioid receptor agonists.4,5,6 Possible strategies to increase methadone's oral bioavailability and duration of drug exposure are numerous. One possible strategy is the concurrent use of a pharmacokinetic enhancer, which is a drug that alters (enhances) the pharmacokinetics of another drug, methadone in this case, leading to increased bioavailability and duration of effect. Previous studies78910 have identified chloramphenicol and fluconazole as drugs that can act as pharmacokinetic enhancers of methadone in dogs, presumably through CYP inhibition. Fluconazole increases methadone's oral bioavailability 98− to 176-fold and duration of exposure at least 12-fold.9,10 Opioid effects, measured as decreased rectal temperature, persisted for approximately 12 hours when an oral formulation of methadone-fluconazole was administered, compared with no effect on rectal temperature when an oral formulation of methadone was administered alone.

Given the promising results of studies9,10 with fluconazole as a pharmacokinetic enhancer of methadone in dogs, we then sought to reduce the potential of misuse and abuse of methadone by people (including theft for the purpose of diversion); methadone intended for use in dogs could be diverted for illicit use. An additional risk of methadone is accidental, possibly life-threatening ingestion by infants or children. Therefore, we developed a formulation that contains naltrexone, primarily a μ-opioid receptor competitive antagonist, to antagonize the opioid effects of methadone when people, but not dogs, ingest this formulation.10 In people, naltrexone has low yet higher oral bioavailability than in dogs, and a portion of naltrexone is consistently metabolized to an active metabolite, 6β-naltrexol, that further antagonizes the opioid effects of methadone. Conversely, in dogs, naltrexone is minimally metabolized to 6β-naltrexol but instead forms the inactive metabolite naltrexone glucuronide.10,11 We have previously9,10 demonstrated that an oral formulation of methadone-fluconazole-naltrexone at a ratio of 1:5:0.25 causes central opioid effects in dogs, as measured by decreased rectal temperature.

Previous studies9,10 also reveal that fluconazole administered 12 hours prior to methadone administration yields measurable opioid effects in dogs. However, whether fluconazole needs to be administered prior to methadone administration or whether fluconazole can be concurrently administered with methadone to increase methadone's oral bioavailability has not yet been determined. Therefore, the purposes of the study reported here were to assess the pharmacokinetics and opioid effects of methadone when administered concurrently with fluconazole and naltrexone (ie, no preadministration of fluconazole) and to assess the pharmacokinetics and opioid effects after administration of multiple doses by means of 2 dosing regimens of methadone-fluconazolenaltrexone.

Materials and Methods

Animals

Twelve 3− to 4-year-old healthy Beaglesa were included, and to ensure equal distribution, dogs were initially blocked by sex and then randomly assigned to 1 of 2 treatments (dosing regimens) of methadone-fluconazole-naltrexone by drawing names from a container. Each group consisted of 4 neutered male and 2 spayed female dogs in a parallel study design. Dogs were confirmed healthy on the basis of history, physical examination, and results of CBC and serum biochemistry analysis. This study was approved by the Institutional Animal Care and Use Committee at Kansas State University.

Procedure

Treatment 1—Dogs (n = 6) were administered an oral formulation of methadone-fluconazolenaltrexone at 6:00 pm on day 1, 8:00 am and 6:00 pm on day 2, and 8:00 am on day 3 (Supplementary Table S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.3.171). The methadone-to-fluconazole-to-naltrexone ratio was 1:5:0.25 mg/kg (doses administered at 6:00 pm on day 1 and 8:00 am on day 2) or 0.5:2.5:0.125 mg/kg (doses administered at 6:00 pm on day 2 and 8:00 am on day 3). Rectal temperature was measured at baseline (time 0) and 20 times thereafter, beginning 1 hour through 70 hours after administration of the first dose. The same rectal thermometer was used for all measurements and was cleaned and disinfected between measurements. Degree of sedation for each dog was assessed by 1 investigator (PC) who was blinded to the administered formulation of methadone-fluconazole-naltrexone using a categorical scale, as follows: none (no apparent sedation), slight (almost normal and able to stand easily but appeared somewhat fatigued, subdued, or somnolent), moderate (able to stand but more often recumbent and appeared sluggish, ataxic, or uncoordinated), profound (unable to rise but showed some awareness of environment, responded to stimuli through body movement, and may have been laterally or sternally recumbent), or unresponsive (in a state of coma or semicoma in which little or no response to stimuli could be elicited and remained in lateral recumbency).10,12

Von Frey measurements, obtained at the same time points as those for rectal temperature, were determined with an electronic deviceb calibrated between 100 and 1,000 g of pressure at the tip as previously described.13,14,15,16 Briefly, the tip of the device was pressed into the carpal pad of each dog until withdrawal movement or vocalization occurred. Simple withdrawal when the tip contacted the carpal pad but before pressure was applied was not recorded as a response. Three von Frey measurements were obtained for the carpal pad of each forelimb at each time point (ie, 6 measurements/dog at each time point, with 6 dogs/time point for a total of 36 measurements/time point). The same investigator (PC) that graded the degree of sedation obtained all von Frey measurements.

Blood samples were obtained at 22 time points between 1 and 70 hours after administration of the first dose and after von Frey measurements were obtained when blood samples and von Frey measurements were required at the same time points on the basis of the study protocol. Blood samples were obtained from aseptically placed jugular catheters with a 3-syringe technique. Briefly, 1 mL of blood was collected into a syringe containing 1 mL of saline (0.9% NaCl) solution with 5 U of heparin/mL. A second syringe was used to collect blood through the catheter, the blood-heparin-saline solution mixture (in the first syringe) was injected into the catheter, and 3 mL of saline solution in a third syringe was injected into the catheter. Three milliliters of blood was collected at each time point and placed in a tube containing lithium heparin. The tube was gently inverted and then placed on ice for up to 4 hours until centrifugation for 10 minutes at 3,000 × g. Plasma was separated and stored at −70°C until analysis. After the plasma was prepared with pass-through plates,c it was analyzed for methadone, fluconazole, naltrexone, β-naltrexol, and naltrexone glucuronide by previously validated methods of ultrahigh-performance liquid chromatographyd with triple quadrupole mass spectrometry.9,10,e

Treatment 2—Dogs (n = 6) were administered methadone-fluconazole-naltrexone at 8:00 am, 12:00 pm, and 6:00 pm on day 1 and 8:00 am on day 2 (Supplementary Table S2, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.3.171). The methadone-to-fluconazole-to-naltrexone ratio was 1:5:0.25 mg/kg (dose administered at 8:00 am on day 1) or 0.5:2.5:0.125 mg/kg (doses administered at 12:00 pm and 6:00 pm on day 1 and 8:00 am on day 2). Rectal temperature was measured at baseline (time 0) and 17 times thereafter, beginning 2 hours through 72 hours after administration of the first dose. The same rectal thermometer was used for all measurements and was cleaned and disinfected between measurements. Degree of sedation was assessed and von Frey measurements were obtained as for treatment 1; however, the last von Frey measurement was 56 hours (vs 70 hours) after administration of the first dose. Blood samples were obtained at baseline (time 0) and at 18 time points thereafter, from 2 through 72 hours after administration of the first dose and after von Frey measurements were obtained when blood samples and von Frey measurements were required at the same time points on the basis of the study protocol. Blood samples were collected and processed as for treatment 1.

Treatment preparation—Size 0 gelatin capsulesf were filled with 1 of 2 doses of methadone-fluconazole-naltrexone. Tablets of each drug were weighed, crushed, and homogenized to a fine powder with a mortar and pestle. Then, the powder was aliquoted by weight into gelatin capsules, and capsules were formulated to be within 95% to 105% of content by weight. One capsule contained 6 mg of methadone (from 5-mg tablets),g 30 mg of fluconazole (from 50− and 200-mg tablets),h and 1.5 mg of naltrexone (from 50-mg tablets),i equivalent to approximately 0.5 mg of methadone/kg (mean; range, 0.43 to 0.58 mg/kg) 2.5 mg of fluconazole/kg (2.17 to 2.91 mg/kg), and 0.125 mg of naltrexone/kg (0.11 to 0.15 mg/kg). Another capsule contained twice the amount of each drug (ie, 12 mg of methadone, 60 mg of fluconazole, and 3 mg of naltrexone, equivalent to approx 1 mg of methadone/kg [mean; range 0.87 to 1.17 mg/kg], 5 mg of fluconazole/kg [4.33 to 5.83 mg/kg], and 0.25 mg of naltrexone/kg [0.22 to 0.29 mg/kg]). However, on the basis of previous studies,9,10 we predicted methadone would accumulate; therefore, methadone doses were decreased from 12 to 6 mg after administration of the second dose (ie, 6 mg administered at 6:00 pm on day 2 and 8:00 am on day 3) for treatment 1 dogs and from 12 to 6 mg after the first dose (ie, 6 mg administered at 12:00 pm and 6:00 pm on day 1 and 8:00 am on day 2) for treatment 2 dogs. Concurrently, fluconazole doses were decreased from 60 to 30 mg and naltrexone doses were decreased from 3 to 1.5 mg to maintain the methadone-to-fluconazole-to-naltrexone ratio (from 1:5:0.25 to 0.5:2.5:0.125 mg/kg).

Pharmacokinetics—Pharmacokinetic data were analyzed by noncompartmental methods with computer software.j The Cmax and tmax were determined directly from the data. Terminal half-life was determined after the last dose with log-linear regression analysisj and included the time points on the terminal slope of the curve. The lowest mean plasma Cmin (trough concentration) of methadone that was associated with significant opioid effects on rectal temperature was determined directly from the data. Plasma concentrations between sampling times were extrapolated on the basis of log-linear regression analysis.k

Statistical analysis

Statistical analyses of pharmacokinetic and pharmacodynamic data were performed with computer software.k Normality and equal variance for rectal temperatures were confirmed with the Shapiro-Wilk test and Spearman rank correlation, respectively. Rectal temperature data were compared within groups by use of a 1-way repeated-measures ANOVA followed by the Holm-Sidak post hoc test for multiple comparisons. von Frey measurements were expressed as percentage change from baseline by dividing the von Frey measurement after drug administration by the mean baseline measurement and multiplying by 100%. Results were compared within group by use of a 1-way repeated-measures ANOVA followed by the Holm-Sidak post hoc test for multiple comparisons with baseline. Values of P < 0.05 were considered significant.

Results

The mean percentage of targeted drug weight in the capsules containing 6 mg of methadone, 30 mg of fluconazole, and 1.5 mg of naltrexone was 100% (range, 97% to 103%). The mean percentage of targeted drug weight in the capsules containing 12 mg of methadone, 60 mg of fluconazole, and 3 mg of naltrexone was 100% (range, 96% to 105%).

Treatment 1 dogs

Mean body weight of treatment 1 dogs was 12.4 kg (range, 10.5 to 13.5 kg). Compared with baseline, rectal temperature was significantly decreased from 2 hours after the first dose through 50 hours (12 hours after the last [fourth] dose), except at 14 and 15 hours (Figure 1). Significant antinociception as measured with the von Frey device occurred at 1, 2, 4, 16, 18, and 28 hours after the first dose (28 hours was 4 hours after the third dose; Figure 2). One dog had a visible lesion (scab) approximately 1 mm in diameter on 1 carpal pad, the application site of the von Frey device, at 46 hours (8 hours after the last [fourth] dose); therefore, data for that dog were limited to 1 (contralateral) carpal pad for the last 4 measurements. Sedation was categorized as mild for 5 dogs and moderate for 1 dog.

Figure 1
Figure 1

Mean ± SD rectal temperature of 6 healthy dogs obtained after oral administration of 1 of 2 dosing regimens (treatment 1) of methadone-fluconazole-naltrexone at time 0 (baseline) and 14, 24, and 38 hours after the first dose (arrows). Decreased temperature from baseline was considered an opioid (methadone) effect. *Value is significantly (P < 0.05) different, compared with baseline.

Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.171

Figure 2
Figure 2

Mean ± SD percentage of baseline von Frey measurements (vF) for the dogs of Figure 1. See Figure 1 for key.

Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.171

Plasma concentrations of methadone, fluconazole, and naltrexone glucuronide were summarized (Table 1; Supplementary Table S3, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.3.171) and graphed (Figures 3 and 4). The mean plasma concentration of methadone was 30.5 ng/mL (range, 1.5 to 59.3 ng/mL) 1 hour after the first dose, and the plasma concentration was quantifiable through 70 hours (32 hours after the last [fourth] dose). The range of mean Cmax was 30.6 to 39.5 ng/mL among the 4 doses, and mean terminal half-life was 9.4 hours after the fourth dose (Table 2). Mean terminal half-life could only be determined for 5 of the 6 dogs because the plasma concentration of methadone in 1 dog had plateaued between 12 and 32 hours after the fourth dose.

Table 1

Mean (range) plasma concentrations of methadone, fluconazole, and naltrexone glucuronide in 6 healthy dogs after oral administration of treatment 1 (dosing regimen) of methadone-fluconazole-naltrexone at a ratio of 1:5:0.25 mg/kg (12:60:3 mg) at 0 and 14 hours and at a ratio of 0.5:2.5:0.125 mg/kg (6:30:1.5 mg) at 24 and 38 hours.

Time (h) Methadone (ng/mL) Fluconazole (μg/mL) Naltrexone glucuronide (ng/mL)
1.0 30.5 (1.5–59.3) 4.21 (1.01–6.24) 71.3 (24.6–94.4)
2.0 38.4 (9.0–68.9) 4.66 (3.28–6.20) 47.6 (30.4–77.6)
4.0 27.2 (6.9–52.4) 4.42 (3.42–5.48) 19.9 (13.7–32.5)
14.0 11.1 (5.8–19.7) 3.53 (2.51–4.28) 14.6 (8.4–27.1)
14.5 31.3 (5.5–125.4) 4.56 (3.23–8.22) 46.9 (9.8–183.6)
15.0 30.1 (6.7–80.5) 5.02 (2.91–8.40) 53.2 (10.2–116.6)
16.0 34.9 (7.7–69.2) 5.92 (3.13–8.11) 56.1 (14.4–94.3)
18.0 30.8 (8.7–50.3) 5.77 (3.37–7.65) 35.5 (19.4–54.9)
20.0 26.3 (14.2–39.4) 5.72 (4.39–6.89) 26.7 (16.0–50.8)
24.0 21.3 (11.7–34.1) 5.73 (3.89–7.80) 28.2 (14.5–57.1)
25.0 21.4 (12.3–36.0) 5.93 (3.57–7.75) 37.7 (15.8–86.2)
26.0 24.2 (14.2–49.3) 6.37 (4.15–7.67) 39.4 (9.9–61.7)
28.0 28.1 (19.4–49.1) 6.84 (4.96–8.48) 43.3 (15.4–99.6)
38.0 26.2 (17.2–44.8) 7.05 (5.63–10.29) 30.3 (15.3–61.3)
38.5 26.7 (15.6–48.5) 7.39 (5.72–10.19) 48.7 (20.8–122.9)
39.0 29.7 (16.2–53.3) 7.89 (6.32–10.77) 43.3 (21.3–79.0)
40.0 34.0 (14.6–59.2) 8.54 (6.40–12.69) 46.2 (23.9–101.3)
42.0 35.5 (18.7–74.9) 8.42 (6.10–11.27) 43.9 (21.1–94.4)
46.0 31.1 (20.3–54.8) 8.01 (5.90–11.16) 30.7 (19.5–40.6)
50.0 27.2 (17.4–50.0) 7.09 (5.98–9.05) 21.5 (10.0–35.7)
62.0 14.8 (6.6–27.7) 5.35 (4.37–6.36) 17.3 (9.2–26.5)
70.0 9.7 (3.3–24.2) 4.38 (2.99–5.92) 19.9 (12.9–42.8)

Blood samples were obtained prior to drug administration when both sample collection and drug administration were scheduled for the same time point.

Figure 3
Figure 3

Mean plasma concentrations of methadone (black circles) and naltrexone glucuronide (white circles) in the dogs of Figure 1. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.171

Figure 4
Figure 4

Mean plasma concentrations of fluconazole after oral administration of 4 doses of 1 of 2 dosing regimens (treatment 1 [circles]; treatment 2 [squares]) of methadone-fluconazole-naltrexone to 12 healthy dogs (6 dogs/treatment). Doses for treatment 1 were administered at 0, 14, 24, and 38 hours and for treatment 2 at 0, 4, 10, and 24 hours.

Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.171

Table 2

Pharmacokinetics of methadone after administration of 1 of 2 dosing regimens (treatments) of methadone-fluconazole-naltrexone to 12 healthy dogs (6 dogs/treatment).

Treatment Cmax (ng/mL) Cmin* (ng/mL) tmax (h) t1/2 (h)
Treatment 1
 Dose 1 31.7 (9.0–68.9) 11.1 (5.8–19.7) 2.2 (2.0–4.0)
 Dose 2 39.5 (19.0–125.4) 21.3 (11.7–34.1) 2.7 (0.5–6.0)
 Dose 3 30.6 (21.4–49.3) 26.2 (17.2–44.8) 3.5 (1.0–14.0)
 Dose 4 36.7 (23.0–74.9) 27.2 (17.4–50.0) 3.0 (0.5–12.0) 9.4 (7.9–12.4)
Treatment 2
 Dose 1 43.5 (17.2–127.2) 42.8 (17.2–91.3) 2.5 (2.0–4.0)
 Dose 2 40.2 (16.9–112.6) 1.0 (1.0–1.0)
 Dose 3 33.1 (13.2–99.8) 28.1 (13.2–58.3) 4.3 (1.0–14.0)
 Dose 4 35.2 (24.1–58.5) 27.0 (21.3–39.7) 4.3 (1.0–12.0) 13.0 (7.3–23.7)

Data are given as mean (range).

The pharmacokinetic parameters were determined after administration of each dose, with the exception of terminal half-life (t1/2), which was determined after the fourth dose on the basis of the data from 5 of 6 dogs. Treatment 1 doses were administered at 0, 14, 24, and 38 hours. Treatment 2 doses were administered at 0, 4, 10, and 24 hours. Blood samples were obtained prior to dosing when sample collection and dose administration were scheduled for the same time point. Cmin* for dose 4 was determined 12 hours after its administration.

— = Not determined.

The lowest mean plasma Cmin of methadone associated with significant effects on rectal temperature was 21.3 ng/mL (Cmin from the second dose in treatment 1). Determined from a log-linear regression model, the time for the plasma concentration to attain 21.3 ng/mL after administration of the first treatment 1 dose was 6.9 hours.

Treatment 2 dogs

The mean body weight of treatment 2 dogs was 12.3 kg (range, 10.3 to 13.9 kg). Rectal temperature was significantly decreased, compared with baseline, from 2 hours after the first dose through 36 hours (12 hours after the last [fourth] dose; Figure 5). Significant antinociception occurred starting 2 hours after the first dose and through 14 hours (Figure 6). One dog had a visible lesion (scab) approximately 1 mm in diameter on 1 carpal pad, the application site of the von Frey device, at 56 hours (32 hours after the last [fourth] dose); therefore, data for that dog were limited to 1 (contralateral) carpal pad for the last measurement. Sedation was categorized as mild for 3 dogs and moderate for 3 dogs.

Figure 5
Figure 5

Mean ± SD rectal temperature of 6 healthy dogs obtained after oral administration of 1 of 2 dosing regimens (treatment 2) of methadone-fluconazole-naltrexone at 0, 4, 10, and 24 hours (arrows). Decreased temperature from baseline was considered an opioid (methadone) effect. *Value is significantly (P < 0.05) different, compared with baseline.

Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.171

Figure 6
Figure 6

Mean ± SD percentage of baseline von Frey measurements (vF) for the dogs of Figure 5. See Figure 5 for key.

Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.171

Plasma concentrations of methadone, fluconazole, and naltrexone glucuronide were summarized (Table 3) and graphed (Figures 4 and 7). The mean plasma concentration of methadone was 52.4 ng/mL (range, 7.7 to 127.2 ng/mL) 2 hours after the first dose, and plasma concentration was quantifiable through 56 hours (32 hours after the last [fourth] dose). The range of mean Cmax was 33.1 to 43.5 ng/mL among the 4 doses, and mean terminal half-life was 13.0 hours after the fourth dose Table 2). Terminal half-life could only be determined for 5 of the 6 dogs because the plasma concentration of methadone for 1 dog had plateaued between 12 and 32 hours after the fourth dose. The mean plasma Cmin for methadone after the first, third, and fourth doses was 42.8 (at 4 hours), 28.1 (at 24 hours), and 27 (at 36 hours [12 hours after the fourth dose]) ng/mL, respectively. No blood sample was obtained to determine the Cmin after the second dose.

Table 3

Mean (range) plasma concentrations of methadone, fluconazole, and naltrexone glucuronide in 6 healthy dogs after oral administration of 1 dosing regimen (treatment 2) of methadone-fluconazole-naltrexone at a ratio of 1:5:0.25 mg/kg (12:60:3 mg) at time 0 and at a ratio of 0.5:2.5:0.125 mg/kg (6:30:1.5 mg) at 4, 10, and 24 hours.

Time (h) Methadone (ng/mL) Fluconazole (μg/mL) Naltrexone glucuronide (ng/mL)
2.0 52.4 (7.7–127.2) 6.18 (4.46–7.96) 62.0 (33.9–84.0)
4.0 42.8 (17.2–91.3) 5.64 (4.69–7.12) 31.6 (11.7–70.2)
5.0 52.6 (16.9–112.6) 5.49 (4.46–6.24) 28.5 (21.5–35.2)
6.0 45.6 (14.5–95.6) 5.59 (4.33–7.07) 40.5 (19.9–77.0)
8.0 39.7 (12.0–100.7) 5.84 (4.16–6.95) 31.6 (18.2–53.3)
10.5 30.0 (13.1–70.5) 5.44 (4.26–7.36) 22.9 (7.4–34.1)
11.0 30.1 (12.4–70.9) 5.83 (4.16–7.27) 22.2 (15.8–32.9)
12.0 32.0 (11.8–65.5) 6.57 (4.95–8.55) 34.7 (18.7–50.5)
14.0 38.8 (12.7–99.8) 6.92 (5.52–8.21) 44.9 (30.5–61.8)
24.0 28.1 (13.2–58.3) 6.43 (5.08–9.19) 25.2 (5.7–36.8)
24.5 27.6 (14.0–57.1) 6.31 (4.97–9.42) 21.5 (5.5–32.6)
25.0 27.4 (14.1–57.9) 5.92 (4.91–8.27) 23.3 (9.7–33.8)
26.0 26.9 (16.6–52.1) 6.24 (5.38–8.01) 28.7 (11.1–42.6)
28.0 35.3 (24.1–58.5) 8.04 (5.32–11.32) 52.6 (6.9–93.1)
32.0 32.2 (23.7–48.6) 7.49 (4.98–9.86) 34.8 (8.8–62.3)
36.0 27.0 (21.3–39.7) 6.88 (4.50–9.47) 28.0 (6.4–38.0)
48.0 19.0 (8.7–35.6) 4.99 (3.60–6.28) 20.9 (11.0–31.0)
56.0 11.8 (3.8–24.0) 4.05 (2.41–5.43) 11.0 (7.1–18.1)

Samples were obtained prior to drug administration when both sample collection and drug administration were scheduled for the same time point.

Figure 7
Figure 7

Mean plasma concentrations of methadone (black squares) and naltrexone glucuronide (white squares) in the dogs of Figure 5. See Figure 5 for remainder of key.

Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.171

Metabolism

The inactive metabolite naltrexone glucuronide was detected in all 12 dogs. Naltrexone (10/240 samples [n = 10/132 for treatment 1 and 0/108 for treatment 2]) and β-naltrexol (5 [0 for treatment 1 and 5 for treatment 2]) were detected at 12 time points. For 10 of those time points, von Frey measurements were obtained concurrently and concentrations of naltrexone and β-naltrexol did not appear to affect von Frey measurements. For 7 of those 10 time points, significant antinociception was present, and for 2 of the 3 time points in which significant antinociception was not present, plasma methadone concentrations were < 21.3 ng/mL.

Discussion

The findings reported here were the first regarding the pharmacokinetics and opioid effects of methadone when coadministered with fluconazole and naltrexone as an oral formulation and dosed multiple times. This study was also the first to assess the pharmacokinetics of an oral formulation of methadone-fluconazole-naltrexone without administration of fluconazole 12 hours before administration of methadone, as has been previously reported.9,10 In those previous studies,9,10 fluconazole administered prior to methadone increased methadone's bioavailability. However, no studies have revealed whether preadministration of fluconazole is necessary to increase methadone's bioavailability or whether bioavailability would increase with coadministration of methadone and fluconazole. The pharmacokinetics and opioid effects documented with both treatments of methadone-fluconazole-naltrexone in the present study indicated that the interaction between methadone and fluconazole occurred rapidly without the need to administer fluconazole prior to administering methadone. Opioid effects, measured as decreased rectal temperature and as antinociception, were present within 2 hours after administration of either treatment.

Antinociceptive effects were noted with each treatment after the first dose but were not maintained throughout the study. Antinociceptive effects may not have been maintained because of the antinociceptive model used. In previous studies13,14,15 of dogs, von Frey measurements were repeatable without evidence of tolerance to the stimuli, learned aversion, or hyperalgesia over a period of 8 to 12 hours. However, in a subsequent study16 of dogs, learned aversion or hyperalgesia was observed when the von Frey device was used for 12 hours, with von Frey measurements significantly lower at 12 hours, compared with baseline. Aversion or hyperalgesia may have occurred in the dogs of the present study and may have resulted in an apparent loss of an antinociceptive effect. This phenomenon has been similarly described with mechanical stimuli in rodents.17 The von Frey device may be limited to short-term use (approx 8 to 12 hours) to evaluate antinociception in dogs. Inclusion of a placebo group may have helped indicate whether aversion or hyperalgesia could develop.

Additionally, 1 dog in each treatment group developed visible lesions on their carpal pads, and, the lesions may have affected the dogs’ responses to the von Frey device (eg, loss of antinociception). Microscopic lesions may have developed on the carpal pads of the other dogs and may have also affected their responses. Additionally, antinociception may have been lost between dose administration times; however, plasma drug concentrations were similar between dose administrations, and the central-mediated hypothermic effects (decreased rectal temperature) of methadone persisted, making loss of antinociception less likely. Although opioid tolerance is an inevitable consequence of repeated opioid administration (and therefore loss of antinociception), tolerance may take several weeks to occur in dogs on the basis of the results of a study18 that included a different antinociceptive model.

Another potential factor for unsustained antinociceptive effects throughout the present study was antagonism of the effects of methadone by naltrexone or β-naltrexol. However, for 7 of the 10 time points in which plasma naltrexone or β-naltrexol was detected, significant antinociception was still present. For 2 of the 3 other time points in which plasma naltrexone or β-naltrexol was detected, significant antinociception was not present and plasma methadone concentrations were < 21.3 ng/mL, suggesting that low methadone concentrations were a contributing factor to the loss of antinociception at those time points.

Other models of antinociception may be useful in dogs for longer duration of testing (vs 8 to 12 hours for the von Frey model). Thermal antinociception has been demonstrated in dogs after administration of various opioids, but thermal antinociception was evaluated after a single dose for an 8-hour period.19 Whether aversion, tolerance, or hyperalgesia would occur beyond 8 hours is unclear. A study20 that included the use of a different thermal antinociception model reveals that antinociception is evident from 48 to 72 hours after dermal application of buprenorphine, suggesting that dogs do not develop aversion, tolerance, or hyperalgesia. Therefore, a validated thermal antinociceptive model that does not result in tissue injury may be better for assessing antinociception for periods > 8 hours. The loss of antinociception to the von Frey device should be interpreted in context of the limitations of the model used in the present study.

No model of antinociception in dogs has been correlated to clinical analgesia, but a model serves as a tool to develop and assess the efficacy of analgesics. Advancing drugs to clinical trials without the use of a model of antinociception or physiologic markers to characterize a drug dosage could result in persistent, uncontrolled pain in a patient versus momentary and reversible nociception in a preclinical study. As such, antinociception testing is a tool to evaluate potential analgesics and develop dosage regimens with a reasonable expectation of achieving success in patients; however, a rescue protocol is needed in clinical trials if the analgesic under study fails.

A decrease in rectal temperature after opioid administration is a centrally mediated opioid effect in dogs. In treatment 2 dogs, rectal temperature was significantly decreased from 2 hours after the first dose through at least 12 hours after the last dose (36 hours total), compared with baseline (prior to treatment). The corresponding mean plasma Cmin for methadone was 42.8, 28.1, and 27 ng/mL after doses 1, 3, and 4, respectively. In treatment 1 dogs, however, rectal temperature was initially significantly decreased (vs baseline) but the decrease was not significant by 14 hours after the first dose, indicating that the opioid effects may have been lost sometime between 4 and 14 hours (last measurement of rectal temperature before 14 hours was at 4 hours), with a mean Cmin of 11.1 ng/mL at 14 hours. Yet, rectal temperature again was significantly decreased at 16 hours after administration of the first dose (2 hours after the second dose) until at least 12 hours after the last dose (from 16 to 50 hours). The mean plasma Cmin for treatment 1 dogs was 21.3, 26.2, and 27.2 ng/mL after doses 2, 3, and 4, respectively, when concurrent significant temperature decreases were present. Therefore, on the basis of these data, a mean plasma Cmin of ≥ 21.3 ng/mL of methadone for either treatment was associated with maintaining significant opioid effects on rectal temperature. On the basis of log-linear extrapolation between 4 and 14 hours after the first dose of treatment 1, 21.3 ng/mL occurred at 6.9 hours, suggesting the second dose should be administered at approximately 6 hours and then every 12 hours to maintain persistent opioid effects throughout the entire dosing interval. By use of this extrapolation, treatment 2 maintained opioid effects when the second dose was administered at 4 hours. However, studies confirming the ideal dosing intervals need to be performed.

Maximum concentration, tmax, and half-life of methadone after administration of the last (fourth) dose of both treatments of methadone-fluconazole-naltrexone were similar. In a previous study,9 fluconazole administered 12 hours prior to methadone (1 mg/kg, PO) achieved a range of methadone Cmax from 25.4 to 45.9 ng/mL, similar to the Cmax (treatment 1, 36.7 ng/mL; treatment 2, 35.2 ng/mL) achieved after administration of the last (fourth) doses of either treatment, with a range of half-life from 8.99 to 9.49 hours, in the present study. In another study,10 methadone (1 mg/kg, PO) administered 12 hours after fluconazole achieved a mean Cmax of 35.1 ng/mL with a half-life of 7.92 hours, and methadone coadministered with naltrexone achieved a Cmax of 33.5 ng/mL and a half-life of 7.09 hours for methadone, similar to the Cmax and half-lives after administration of the last (fourth) doses of both treatments in the present study.

We predicted methadone would accumulate in the plasma with administration of multiple doses. Therefore, for treatment 1, we decreased the dose of methadone from 1 mg/kg for the first and second doses to 0.5 mg/kg for the third and fourth doses and, for treatment 2, from 1 mg/kg for the first dose to 0.5 mg/kg for the second, third, and fourth doses. The mean plasma Cmin for treatment 1 was 11.1, 21.3, 26.2, and 27.2 ng/mL after doses 1, 2, 3, and 4 (Cmin measured 12 hours after the fourth dose), respectively. The mean plasma Cmin for treatment 2 was 42.8, 28.1, and 27.0 ng/mL after doses 1, 3, and 4 (Cmin measured 12 hours after the fourth dose), respectively. The plasma Cmin for both treatments suggested that decreasing the dose was appropriate to minimize drug accumulation and potentially excessive opioid effects (eg, sedation and profound hypothermia).

Fluconazole is a safe and effective antifungal in dogs, but for the present study, it was repurposed as a pharmacokinetic enhancer.21,22 Our previous studies9,10 included the administration of fluconazole at least 12 hours prior to the administration of methadone to allow fluconazole to inhibit CYP enzymes that are responsible for the metabolism of methadone. In the present study, however, opioid effects were noted after administration of the first dose of the combination of methadone-fluconazole-naltrexone, suggesting that fluconazole administration does not need to precede methadone administration for fluconazole to exert its effects on CYP enzymes. The mean fluconazole plasma concentration at 1 hour (4.21 μg/mL) exceeded the reported23 in vitro half maximal inhibitory concentration of fluconazole for canine CYP2B11 (0.12 μg/mL) by approximately 35 times, suggesting that inhibitory concentrations of fluconazole were rapidly attained, assuming that plasma fluconazole concentrations were related to the in vitro inhibitory concentration. In vitro studies24,25 also reveal that fluconazole is a reversible competitive-noncompetitive inhibitor of CYP in people. If this is also true for canine CYP2B11, this would confirm that the rapid enhancement effect of fluconazole on methadone is directly related to fluconazole's observed pharmacokinetic profile, unlike irreversible inhibitors for which plasma drug concentrations may not directly reflect the amount of inhibited enzyme. One potential benefit for fluconazole pretreatment is to accumulate fluconazole to further inhibit CYP2B11; however, the modest increase in Cmax in the previous studies9,10 suggests little added benefit of pretreatment.

With the inclusion of fluconazole in a formulation of methadone, a concern exists, as with any antimicrobial, for the selection of resistant organisms. Selection of resistant organisms with orally administered fluconazole is expected to be limited to commensal organisms such as Malassezia spp and Candida spp. Administered antifungals are not expected to select for antifungal-resistant environmental pathogens such as Aspergillus spp, Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum because agricultural use of antifungals is thought to be the primary driver of antifungal resistance.26,27,28,29 We anticipate that the clinical use of methadone-fluconazole-naltrexone will be for the amelioration of signs of acute pain associated with surgery or trauma. Short durations (1 to 3 days) of fluconazole would likely be administered, and we therefore speculate that selection of antifungal-resistant commensal fungal organisms would be minor. In people, fluconazole treatment > 14 days’ duration resulted in a significantly increased risk of infection with fluconazole-resistant Candida spp.30 However, specific data for dogs regarding resistance selection of fungal organisms and the time frame in which it occurs are lacking. Studies assessing the effects of methadone-fluconazole-naltrexone would be needed to determine whether the amount of fluconazole contained within a formulation of methadone-fluconazole-naltrexone would select for fluconazole-resistant fungal organisms.

Naltrexone was included in these formulations of methadone-fluconazole as a deterrent to human opioid abuse and misuse.31 Currently, some veterinarians may not be prescribing opioids for dogs with signs of pain because of concerns of diversion or abuse by pet owners. A recent survey32 of veterinarians in South Dakota indicates that nearly 70% have modified the amount, duration, or dose of prescribed opioids because of pet owner characteristics (ie, suspicious behavior), independent of the degree of patient pain. In the dogs of the present study, however, opioid effects were maintained after administration of either treatment of methadone-fluconazole-naltrexone, most likely secondary to naltrexone's low bioavailability and metabolism to the inactive metabolite naltrexone glucuronide. Inclusion of naltrexone in a formulation of methadone-fluconazole may increase veterinarians’ comfort in prescribing methadone (as a methadone-fluconazole-naltrexone formulation vs other opioids) for analgesia and simultaneously reduce the public health risks of misuse, abuse, or accidental ingestion. Development of a safe and effective opioid formulation (eg, methadone-fluconazole-naltrexone) for dogs with a deterrent to human misuse and abuse may better the management of pain in dogs.

The present study documented rapid opioid effects, as determined by means of rectal temperature and an antinociception model, in dogs after oral administration of methadone-fluconazole-naltrexone after the first dose and multiple-dose regimens. Fluconazole administration hours before methadone administration was not needed to maintain plasma methadone concentrations associated with antinociception. Opioid effects were noted despite inclusion of the μ-opioid receptor competitive antagonist naltrexone. Naltrexone was included to decrease potential opioid abuse and misuse and consequences of inadvertent opioid ingestion by people. Studies of the clinical effects in dogs and the antiabuse potential in people of an oral formulation of methadone-fluconazole-naltrexone are needed.

Acknowledgments

Funded by the College of Veterinary Medicine, Mark Derrick Fund, Veterinary Research Scholars Program, Veterinary Student Mentoring Program (Department of Clinical Sciences), and McNair Scholars Program, Kansas State University. The Phoenix software license was provided by Certara USA, Inc as a part of the company's Academic Centers of Excellence program.

Kansas State University has applied for a patent covering the intellectual property reported in this manuscript.

Presented in abstract form at the Annual Forum of the American College of Veterinary Internal Medicine, Phoenix, June 2019.

The authors thank Dr. Hyun Joo for determining the plasma drug concentrations.

Abbreviations

Cmax

Maximum observed concentration

Cmin

Minimum observed concentration

CYP

Cytochrome P450

tmax

Time to maximum concentration

Footnotes

a.

Marshall Bioresources, North Rose, NY.

b.

IITC Life Science Inc, Woodland Hills, Calif.

c.

Ostro pass-through sample preparation plate, Waters Corp, Milford, Mass.

d.

Acquity Prominence UPLC, Waters Corp, Milford, Mass.

e.

TQD, Waters Corp, Milford, Mass.

f.

Capsuline, Pompano Beach, Fla.

g.

Elite Pharmaceuticals Inc, Northvale, NJ.

h.

Glenmark Pharmaceuticals Ltd, Colvale Bardez, Goa, India.

i.

Mallinckrodt Inc, Hazelwood, Mo.

j.

Phoenix, 64-bit version, Certara Inc, Princeton, NJ.

k.

SigmaPlot, version 12.5, Systat Software Inc, San Jose, Calif.

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Contributor Notes

Dr. Locuson's present address is Agios Pharmaceuticals, Cambridge, MA 02139.

Dr. Cho's present address is the VCA Veterinary Care Animal Hospital and Referral Center, Albuquerque, NM 87111.

Address correspondence to Dr. Butch KuKanich (kukanich@ksu.edu).
  • Figure 1

    Mean ± SD rectal temperature of 6 healthy dogs obtained after oral administration of 1 of 2 dosing regimens (treatment 1) of methadone-fluconazole-naltrexone at time 0 (baseline) and 14, 24, and 38 hours after the first dose (arrows). Decreased temperature from baseline was considered an opioid (methadone) effect. *Value is significantly (P < 0.05) different, compared with baseline.

  • Figure 2

    Mean ± SD percentage of baseline von Frey measurements (vF) for the dogs of Figure 1. See Figure 1 for key.

  • Figure 3

    Mean plasma concentrations of methadone (black circles) and naltrexone glucuronide (white circles) in the dogs of Figure 1. See Figure 1 for remainder of key.

  • Figure 4

    Mean plasma concentrations of fluconazole after oral administration of 4 doses of 1 of 2 dosing regimens (treatment 1 [circles]; treatment 2 [squares]) of methadone-fluconazole-naltrexone to 12 healthy dogs (6 dogs/treatment). Doses for treatment 1 were administered at 0, 14, 24, and 38 hours and for treatment 2 at 0, 4, 10, and 24 hours.

  • Figure 5

    Mean ± SD rectal temperature of 6 healthy dogs obtained after oral administration of 1 of 2 dosing regimens (treatment 2) of methadone-fluconazole-naltrexone at 0, 4, 10, and 24 hours (arrows). Decreased temperature from baseline was considered an opioid (methadone) effect. *Value is significantly (P < 0.05) different, compared with baseline.

  • Figure 6

    Mean ± SD percentage of baseline von Frey measurements (vF) for the dogs of Figure 5. See Figure 5 for key.

  • Figure 7

    Mean plasma concentrations of methadone (black squares) and naltrexone glucuronide (white squares) in the dogs of Figure 5. See Figure 5 for remainder of key.

  • 1.

    KuKanich B. Outpatient oral analgesics in dogs and cats beyond nonsteroidal antiinflammatory drugs: an evidence-based approach. Vet Clin North Am Small Anim Pract 2013;43:11091125.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Adler MW, Geller EB, Rosow CE, et al. The opioid system and temperature regulation. Annu Rev Pharmacol Toxicol 1988;28:429449.

  • 3.

    Vaupel DB, Jasinski DR. l-alpha-acetylmethadol, l-alpha-acetyl-N-normethadol and l-alpha-acetyl-N,N-dinormethadol: comparisons with morphine and methadone in suppression of the opioid withdrawal syndrome in the dog. J Pharmacol Exp Ther 1997;283:833842.

    • Search Google Scholar
    • Export Citation
  • 4.

    Garrett ER, Derendorf H, Mattha AG. Pharmacokinetics of morphine and its surrogates. VII: high-performance liquid chromatographic analyses and pharmacokinetics of methadone and its derived metabolites in dogs. J Pharm Sci 1985;74:12031214.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    KuKanich B, Borum SL. The disposition and behavioral effects of methadone in Greyhounds. Vet Anaesth Analg 2008;35:242248.

  • 6.

    Kukanich B, Lascelles BD, Aman AM, et al. The effects of inhibiting cytochrome P450 3A, p-glycoprotein, and gastric acid secretion on the oral bioavailability of methadone in dogs. J Vet Pharmacol Ther 2005;28:461466.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Kukanich B, Kukanich KS, Rodriguez JR. The effects of concurrent administration of cytochrome P-450 inhibitors on the pharmacokinetics of oral methadone in healthy dogs. Vet Anaesth Analg 2011;38:224230.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    KuKanich B, KuKanich K. Chloramphenicol significantly affects the pharmacokinetics of oral methadone in Greyhound dogs. Vet Anaesth Analg 2015;42:597607.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    KuKanich B, KuKanich K, Rankin D, et al. The effect of fluconazole on oral methadone in dogs. Vet Anaesth Analg 2019;46:501509.

  • 10.

    KuKanich B, KuKanich K, Rankin DC, et al. Pharmacokinetics and pharmacodynamics of a novel analgesic with a deterrent to human opioid abuse (methadone-fluconazole-naltrexone) after oral administration in dogs. Am J Vet Res 2020;81:656664.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Garrett ER, el-Koussi A el-D. Pharmacokinetics of morphine and its surrogates V: naltrexone and naltrexone conjugate pharmacokinetics in the dog as a function of dose. J Pharm Sci 1985;74:5056.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Martinez SA, Wilson MG, Linton DD, et al. The safety and effectiveness of a long-acting transdermal fentanyl solution compared with oxymorphone for the control of postoperative pain in dogs: a randomized, multicentered clinical study. J Vet Pharmacol Ther 2014;37:394405.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    KuKanich B, Lascelles BD, Papich MG. Assessment of a von Frey device for evaluation of the antinociceptive effects of morphine and its application in pharmacodynamic modeling of morphine in dogs. Am J Vet Res 2005;66:16161622.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    KuKanich B, Lascelles BD, Papich MG. Use of a von Frey device for evaluation of pharmacokinetics and pharmacodynamics of morphine after intravenous administration as an infusion or multiple doses in dogs. Am J Vet Res 2005;66:19681974.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Kukanich B, Papich MG. Pharmacokinetics and antinociceptive effects of oral tramadol hydrochloride administration in Greyhounds. Am J Vet Res 2011;72:256262.

    • Crossref
    • Search Google Scholar
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