Materials and Methods

Animals—The study was approved by the Institutional Animal Care and Use Committee at Kansas State University. Six healthy Greyhounds, 3 males and 3 females, were included in the study. The dogs ranged in age from 3 to 5 years and in weight from 28.5 to 38.5 kg.

Treatments—Treatments were administered in 2 phases. The first treatment phase consisted of morphine sulfate^a (0.5 mg/kg, IV). The second treatment phase, ≤ 7 days after the first treatment phase, consisted of ketoconazole tablets (400 mg, PO)^b administered at 120, 96, 72, 48, 24, and 12 hours and from 1 to 2.5 hours prior to morphine sulfate (0.5 mg/kg, IV) administration. Ketoconazole was administered with approximately 2 tablespoons (approx 30 mL) of peanut butter. Morphine was administered through an aseptically placed cephalic catheter^c during a 1-minute period.

Pharmacodynamics (behavioral assessment)— Animals were observed throughout the study period. Sedation was categorically assessed as none, mild, moderate, or heavy. Episodes of vomiting, panting, and dysphoria were also monitored throughout the study.

Sample collection—Jugular catheters^d were placed aseptically prior to drug administration to facilitate blood sample collection. Blood samples of 9 mL/time point were obtained prior to drug administration and at 10, 20, and 30 minutes and 1, 2, 3, 4, 6, and 8 hours after morphine administration and placed into tubes containing lithium heparin. Jugular catheters were flushed with 3 mL of saline (0.9% NaCl) solution after each sample collection to maintain catheter patency. Blood samples were centrifuged at 3,000 × g for 10 minutes at ambient temperature (approx 21°C), and the plasma was separated and stored frozen at −70°C prior to analysis.

Morphine analyses—Plasma morphine concentrations were determined by liquid chromatography with mass spectrometry by use of electrospray ionization^e according to published methods,¹³ with the following modifications. The mobile phase consisted of 88% citric acid (0.1% wt/vol) and 12% acetonitrile with a flow rate of 0.3 mL/min. Separation was achieved by use of a phenyl column^f maintained at ambient temperature (approx 21°C). Hydromorphone d3^g (0.1 mL, 50 ng/mL) was added to each sample as an internal standard. Sample preparation consisted of adding 0.1M borate buffer to 1 mL of plasma and 0.1 mL of internal standard and vortexing. The plasma mixture then underwent solidphase extraction. Solid-phase extraction cartridges^h were conditioned with methanol (1 mL) and deionized water (1 mL), the plasma mixture was loaded, and then solid-phase extraction cartridges were rinsed with deionized water (1 mL) and eluted with methanol (1 mL). The eluate was evaporated, reconstituted with 0.2 mL of 15% methanol, and then filtered with a centrifugal filter.ⁱ The injection volume was 50 μL.

A plasma standard curve was constructed daily by fortifying untreated plasma with morphine^g at 0 ng/mL and from 1 to 200 ng/mL. The plasma standard curve was accepted if the coefficient of determination was > 0.99 and predicted concentrations were within 15% of the actual concentration. The accuracy (deviation from actual value) of the assay as determined on replicates of 5 each for 1, 20, and 200 ng/mL was within 5 ± 1% of the actual value. The coefficient of variation was 6 ± 2%.

Ketoconazole analysis—Plasma ketoconazole concentrations were determined by liquid chromatography with mass spectrometry by use of electrospray ionization. The mobile phase consisted of 65% trifluoroacetic acid (0.02% solution) and 35% acetonitrile with a flow rate of 0.3 mL/min. Separation was achieved by use of a C-18 column^j maintained at ambient temperature (approx 21°C). Fluconazole^k (50 μg/mL) was added to each sample as an internal standard. Sample preparation consisted of mixing 0.5 mL of plasma with 0.05 mL of internal standard and vortexing. The plasma mixture then underwent solid-phase extraction. Solid-phase extraction cartridges^l were conditioned with methanol (1 mL) and deionized water (1 mL), the plasma mixture was loaded, and then solid-phase extraction cartridges were rinsed with deionized water (1 mL) and eluted with methanol (1 mL). The eluate was diluted 1:1 with deionized water and directly injected with an injection volume of 25 ML. The qualifying ion for ketoconazole had a mass-to-charge ratio of 531, whereas the product ion (mass-to-charge ratio, 489) was used for quantification. The qualifying ion for fluconazole had a mass-tocharge ratio of 306, whereas the product ion (mass-tocharge ratio, 238) was used for quantification.

A plasma standard curve was constructed daily by fortifying untreated plasma with ketoconazole^m at 0 μg/mL and from 0.1 to 50 μg/mL. The plasma standard curve was accepted if the coefficient of determination was ≤ 0.99 and predicted concentrations were within 15% of the actual concentration. The accuracy of the assay as determined on replicates of 5 each for 2, 10, and 20 μg/mL was within 7 ± 1% of the actual value. The coefficient of variation was 6 ± 2%.

Pharmacokinetic analysis—Pharmacokinetic variables were estimated by use of a noncompartmental analysis with computer software.ⁿ The variables determined were the area under the curve from time 0 to infinity by use of the linear trapezoidal rule, area under the first moment curve from time 0 to infinity, Cl_P, apparent volume of distribution at steady state, apparent volume of distribution during the elimination phase, first-order terminal rate constant, t_½ λ_z, and mean residence time by use of noncompartmental analysis. The concentration at time 0 was calculated by use of loglinear regression with the first 2 time points.

Statistical analysis—Pharmacokinetic variables estimated for morphine and morphine administered with ketoconazole were compared by use of computer software^o; the Mann-Whitney rank sum test was used because the distribution of the data was not normal. The significance level was set a priori at a value of P < 0.05. Descriptive statistics of the pharmacokinetic variables include the mean, median, and 25th and 75th percentiles.

Results

In terms of adverse effects, 1 dog vomited, 3 dogs had episodes of panting, and all dogs were mildly to moderately sedated after administration of morphine alone. Administration of morphine with ketoconazole resulted in 3 dogs vomiting, 4 dogs panting, and all dogs being mildly to moderately sedated. No signs of dysphoria were observed during either crossover of the study. The mean ± SEM amount of ketoconazole administered was 12.7 ± 0.6 mg/kg (range, 10.4 to 14.0 mg/mg).

The plasma concentrations of morphine in Greyhounds decreased rapidly after IV administration (Figure 1; Table 1). No significant differences were found in any of the pharmacokinetic variables of morphine between morphine administered alone and morphine administered with ketoconazole (Table 2). The mean t_½ λ_z was 1.28 hours in the morphine group and 1.42 hours in the morphine with ketoconazole group (P = 0.394). The Cl_P of morphine was rapid at 32.55 mL/min/kg in the morphine group and 26.72 mL/min/kg in the morphine with ketoconazole group (P = 0.180). The apparent volume of distribution during the elimination phase was 3.64 L/kg for the morphine group and 3.26 L/kg for the morphine with ketoconazole group (P = 0.937).

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Mean ± SEM plasma morphine concentrations in 6 healthy Greyhounds following morphine sulfate (0.5 mg/kg, IV) administration and morphine sulfate (0.5 mg/kg, IV) administration after treatment with ketoconazole.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.664

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Plasma ketoconazole concentrations were high, with mean concentrations between 15.65 and 21.19 μg/mL during the study period from 10 minutes to 8 hours after morphine administration (Figure 2; Table 3). The t_¹/2 L_z of ketoconazole could not be determined accurately because of the prolonged elimination and the relatively short period during which plasma samples were obtained (up to 8 hours after morphine administration).

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Mean ± SEM plasma ketoconazole concentrations in 6 healthy Greyhounds, where time is time after morphine administration.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.664

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Discussion

Effects of morphine in Greyhounds after IV administration of morphine at 0.5 mg/kg in the present study were similar to those of other reports.^5–7 Pharmacokinetic variables of morphine in Greyhounds are also similar to reported^1–7 values for other dog breeds.

Ketoconazole administered for 5 days resulted in no significant differences in the pharmacokinetic variables of morphine in dogs. Although results of an in vitro study⁸ revealed an inhibition of morphine glucuronide formation, concentrations of ketoconazole required to inhibit metabolism were high, exceeding 80 μg/mL. Therefore, it was not expected that morphine elimination would be significantly affected in dogs when clinically relevant ketoconazole amounts were administered.

Although a slight increase in the t_½ λ_z and decrease in the Cl_P were observed in the ketoconazole-treated dogs, no significant difference was found. The lack of a significant decrease in the elimination of morphine occurred despite the high concentrations of ketoconazole (15.65 to 21.19 μg/mL) achieved during the study period. It is unlikely that administration of clinically relevant amounts of ketoconazole will have an effect on the elimination of morphine in healthy dogs. However, dogs with preexisting liver disease or decreased cardiac output may be more prone to a ketoconazole-induced decrease in the elimination of morphine.

The pharmacokinetics of morphine have been extensively studied in dogs^1–7; however, our report is the first in Greyhounds. It is difficult to make comparisons between multiple studies as a result of differences in study design, analytical methods, pharmacokinetic calculations, housing conditions, ages, weights, health status, and treatment conditions. However, the t_½ λ_z of morphine in Greyhounds, 1.28 hours, in our study was in the range previously reported for other dog breeds: 0.9 hours in Beagles,⁷ 1.2 hours in mixed-breed dogs,¹ and up to 1.6 hours in Beagles.⁵ The volume of distribution (area method) in Greyhounds was 3.64 L/kg, which is within the range previously reported for Beagles (3.6 to 4.6 L/kg).^6,7 The Cl_P of morphine in Greyhounds (32.55 mL/min/kg) was lower than reported values for Beagles (45.1 to 63 mL/min/kg).^6,7 It was not determined whether the difference reflects a true difference in the Cl_P of morphine or is the result of different study designs, analytical methods, and pharmacokinetic analyses. To assess whether a true difference exists, a crossover study including Greyhounds and another dog breed would have to be conducted.

Pharmacodynamics, as assessed by behavioral observations, were similar between the treatment groups in our study and similar to findings of other reports^5–7 on morphine administration in dogs. Morphine administered to Beagles at 0.5 mg/kg resulted in sedation and dysphoria, with 4 of the dogs vomiting.⁶ The Greyhounds were calm, quiet, and easily handled, displaying no signs of dysphoria during either phase of our study. Pharmacodynamics were similar in Greyhounds administered morphine alone and those administered the combination of morphine and ketoconazole, which suggests that p-glycoprotein inhibition has minimal effects on the pharmacodynamics of IV morphine. These results are similar to those in humans in which inhibition of p-glycoprotein resulted in no significant pharmacokinetic or pharmacodynamic differences in morphine.¹²

High and prolonged plasma concentrations of ketoconazole were an unexpected finding in our study. Plasma concentrations of ketoconazole that were achieved were higher than expected following PO administration of ketoconazole at 12.7 ± 0.6 mg/kg (mean ± SEM). In another study,¹⁴ mongrel dogs received ketoconazole at 19.5 to 25.2 mg/kg, which resulted in a maximum plasma concentration of 17.4 ± 16.7 μg/mL and a t_½ λ_z of 1.7 ± 1.7 hours.¹⁴

Peak plasma concentrations of ketoconazole in our study were similar to other study findings,¹⁴ despite the fact that the amount of ketoconazole administered was approximately 50% less. Increased plasma concentrations and prolonged elimination of ketoconazole in Greyhounds may indicate that Greyhounds are poor metabolizers of ketoconazole.

Ketoconazole in humans is metabolized by oxidation and degradation of the imidazole and piperazine rings, oxidative O-dealkylation, and aromatic hydroxy lation.¹⁵ However, the specific enzymes responsible for the metabolism have not been reported and neither have the metabolic pathways in dogs. Treatment with the cytochrome P-450 inducers rifampin, rifampicin, phenytoin, and carbamazepine resulted in decreased plasma ketoconazole concentrations in humans, suggesting that cytochrome P-450 enzymes metabolizing ketoconazole were induced.^16,17

Pharmacokinetics of ketoconazole could not be accurately determined for Greyhounds in the current study. As a result of the prolonged elimination and relatively short blood sample collection period (8 hours), an accurate assessment of the pharmacokinetics and plasma profile could not be determined. The purpose of the study was not to assess the pharmacokinetics of ketoconazole; therefore, the current study design was not able to determine the pharmacokinetics of ketoconazole in Greyhounds. Further studies assessing the pharmacokinetics of ketoconazole in Greyhounds are warranted.

Results of a study¹⁸ of barbiturate anesthetics (thiopental, thiamylal, and methohexital) indicate that Greyhounds are poor metabolizers, resulting in the prolonged anesthetic effects. Pretreatment of Greyhounds with phenobarbital, which induced cytochrome P-450 metabolism, resulted in similar pharmacokinetics and pharmacodynamics of thiopental in Greyhounds as reported for non-Greyhounds.¹⁹ Ketoconazole may be poorly metabolized, similar to thiopental; however, further studies are needed to confirm the metabolism and pharmacokinetics of ketoconazole in Greyhounds.

Differences in the pharmacokinetics of propofol have been described for Greyhounds in comparison to mixed-breed dogs, and potentially, a difference exists in the pharmacokinetics of celecoxib as well.^20–22 A genetic polymorphism in the metabolism of celecoxib, a cytochrome P450 2D substrate, has been described for Beagles, with approximately half of the Beagles classified as extensive metabolizers and the other Beagles classified as poor metabolizers.²⁰ A separate pharmacokinetic study²¹ of celecoxib in Greyhounds resulted in findings similar to those for the poor-metabolizer Beagles. The pharmacokinetics of propofol, a cytochrome P-450 2B substrate,²² were significantly different in Greyhounds, compared with mixed-breed dogs.²³ The Greyhounds had a significantly decreased volume of distribution and Cl_P but a similar t_½ λ_z. Finally, results of a study²⁴ that examined the elimination of antipyrine, a marker for cytochrome P-450-mediated metabolism, in Beagles and Greyhounds revealed that Greyhounds have a significantly slower Cl_P in comparison to Beagles.

In conclusion, ketoconazole had no significant effect on the elimination of morphine or the behavioral effects of morphine in healthy Greyhounds. However, plasma ketoconazole concentrations were higher than expected, and the elimination of ketoconazole was slower than reported values for mixed-breed dogs.