The effects of concurrent administration of cytochrome P-450 inhibitors on the pharmacokinetics of oral methadone in healthy dogs

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1 Veterinary Anaesthesia and Analgesia, 2011, 38, doi: /j x RESEARCH PAPER The effects of concurrent administration of cytochrome P-450 inhibitors on the pharmacokinetics of oral methadone in healthy dogs Butch KuKanich*, Kate S KuKanich & Jessica R Rodriguez* *Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA Correspondence: Butch Kukanich, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA. kukanich@ksu.edu Abstract Objective The objective was to examine the effects of inhibiting cytochrome P450 (CYP) on the pharmacokinetics of oral methadone in dogs. Study design Prospective non-randomized experimental trial. Animals Six healthy Greyhounds (three male and three female). Methods The study was divided into two phases. Oral methadone (mean = 2.1 mg kg )1 PO) was administered as whole tablets in Phase 1. In Phase 2 oral methadone (2.1 mg kg )1 PO) was administered concurrently with ketoconazole (13.0 mg kg )1 PO q 24 hours), chloramphenicol (48.7 mg kg )1 PO q 12 hours), fluoxetine (1.3 mg kg )1 PO q 24 hours), and trimethoprim (6.5 mg kg )1 PO q 24 hours). Blood was obtained for analysis of methadone plasma concentrations by liquid chromatography with mass spectrometry. The maximum plasma concentration (C max ), time to C max (T max ), and the area under the curve from time 0 to the last measurable time point above the limit of quantification of the analytical assay (AUC 0 LAST ) were compared statistically. Results The C max of methadone was significantly different (p = 0.016) for Phase 1 (5.5 ng ml )1 ) and Phase 2 (171.9 ng ml )1 ). The AUC 0 LAST was also significantly different (p = 0.004) for Phase 1 (13.1 hour ng ml )1 ) and Phase 2 ( hour ng ml )1 ). Conclusion and clinical relevance Concurrent administration of CYP inhibitors with methadone significantly increased the area under the curve and plasma concentrations of methadone after oral administration to dogs. Further studies are needed assessing more clinically relevant combinations of methadone and CYP inhibitors. Keywords CYP, dog, inhibition, metabolism, methadone, pharmacokinetics. Introduction Opioids are routinely used in the perioperative period due to their analgesic, sedative, and anxiolytic effects (Wagner 2002). However, the use of opioids in outpatients is limited due to their low oral bioavailability, primarily from pre-systemic metabolism in the intestines and liver, often referred to as first pass metabolism (Riviere 1999). In addition, large variability of the oral bioavailability of opioids indicates that simple dose adjustments would be inappropriate (KuKanich et al. 2005b). A strategy to increase the oral bioavailability of drugs subject to first pass metabolism is to concurrently administer an inhibitor of metabolism, as is the case with the combination of ketoconazole (an inhibitor) and 224

2 cyclosporine (a substrate) in dogs (Myre et al. 1991). Concurrent administration of an inhibitor with a substrate may also decrease the variability of drug absorption. In one study, the percent variability of the area under the concentration curve for cyclosporine in seven dogs was approximately 30%, but decreased to approximately 12% when cyclosporine was combined with ketoconazole (Myre et al. 1991). Methadone is a synthetic opioid marketed as a racemic mixture. Methadone acts as a l opiate receptor agonist, and also variably acts as an N-methyl-D-aspartate (NMDA) receptor antagonist and adrenergic (alpha-2) agonist (Codd et al. 1995; Gorman et al. 1997). Objective measurements of methadone s analgesic effects have not been described in dogs, but in humans plasma concentrations of methadone between 33 and 59 ng ml )1 have been associated with analgesia in acute pain (Gourlay et al. 1982, 1986). The pharmacokinetics of oral methadone have been incompletely described in dogs (KuKanich et al. 2005a). Administration of 2 mg kg )1 PO as a single dose failed to achieve plasma drug concentrations above the limit of quantification of the analytical assay (20 ng ml )1 ). Co-administration of the cytochrome P450 3A12 (CYP3A12) inhibitor ketoconazole (10 mg kg )1 PO q 12 hours for two doses) resulted in no detectable methadone concentrations in 5/6 dogs, but one of the dogs consistently had measurable concentrations of methadone (KuKanich et al. 2005a). The detectable concentrations were presumably due to either inhibited methadone metabolism or increased absorption. The reason for only one dog being affected by ketoconazole administration is unclear. The same study also administered methadone with the human CYP2C19 inhibitor and acid suppressor omeprazole (1 mg kg )1 q 12 hours for five doses), which produced no detectable methadone concentrations. The metabolic pathways of methadone have not been described in dogs. In humans, multiple cytochrome P450s (CYPs) have been identified in the metabolism of methadone. In vitro studies using human liver microsomes identified CYP3A4 and CYP2C8 as the predominant methadone metabolizing CYPs, but CYP2D6 was also involved in methadone metabolism (Wang & DeVane 2003). More recently, in vitro and in vivo studies in humans have suggested that CYP2B6 has a prominent role in the metabolism of methadone (Totah et al. 2008). Ketoconazole is an antifungal drug, which inhibits CYP3A12 in dogs (Lu et al. 2005). Chloramphenicol is a CYP2B11 inhibitor in dogs (Hay Kraus et al. 2000). Fluoxetine and its metabolite, norfluoxetine, are primarily inhibitors of human CYP2D6 (Crewe et al. 1992) with some additional effects as inhibitors of human CYP2C9 (Schmider et al. 1997). The effects of fluoxetine and norfluoxetine on canine CYPs have not been described. Trimethoprim is an inhibitor of human CYP2C8 (Wen et al. 2002), but the effects of trimethoprim on canine CYPs have not been described. The purpose of this study was to assess the effects of concurrent administration of ketoconazole, chloramphenicol, fluoxetine, and trimethoprim on the pharmacokinetics of oral methadone in healthy dogs. The hypothesis was that co-administration of multiple CYP inhibitors would increase the plasma methadone concentrations after oral methadone administration in dogs. Material and methods The Institutional Animal Care and Use Committee at the University approved the study. Six healthy Greyhound dogs were enrolled in the study including three neutered males and three females. The dogs were assessed as healthy based on prior medical history, complete blood counts, serum chemistry panels, and physical exams. The dogs ranged in age from 2 to 4 years and weight ranged from 26.0 to 41.6 kg. The study consisted of a non-randomized block design. In Phase 1, all dogs were first administered methadone HCL as whole tablets (Mallinckrodt, MO, USA) at a targeted dose of 2 mg kg )1. Blood was collected from an aseptically placed jugular catheter prior to methadone administration and at 15, 30, and 45 minutes and 1, 2, 4, 6, 8, and 12 hours after drug administration. Plasma was separated by centrifugation (15 minutes at 3000 g) and stored frozen at )70 C until analysis. A period of at least 21 days separated Phase 1 and Phase 2. In Phase 2, all dogs were administered CYP inhibitors followed by methadone HCL at a targeted dose of 2 mg kg )1 PO. The CYP inhibitors administered were ketoconazole (Pliva, NY, USA), at a targeted dose of 10 mg kg )1 PO q 24 hours for three doses, chloramphenicol (Viceton; Bimeda, IL, USA), at a targeted dose of 50 mg kg )1 PO q 12 hours for four doses, fluoxetine (Pliva) at a targeted dose of 1 mg kg )1 PO q 24 hours 3 doses, and trimethoprim (Watson, CA, USA) at a targeted Ó 2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38,

3 dose of 5 mg kg )1 PO q 24 hours for three doses. These drugs were administered concurrently on the last dose. One hour after the last dose of the CYP inhibitors, methadone was administered at a targeted dose of 2 mg kg )1 PO. Blood was collected from an aseptically placed jugular catheter prior to methadone administration and at 15, 30, and 45 minutes and 1, 2, 4, 6, and 8, 12, 20, and 32 hours after drug administration. Plasma was separated by centrifugation (15 minutes at 3000 g) and stored frozen at )70 C until analysis. Plasma concentrations of methadone were determined using liquid chromatography (Shimadzu Prominence; Shimadzu Scientific Instruments, MD, USA) with mass spectrometry (API 2000; Applied Biosystems, CA, USA). The mobile phase consisted of A: acetonitrile and B: 0.1% formic acid at a flow rate of 0.4 ml minute )1. A mobile phase consisted of 75% B from 0 to 1 minutes with a linear gradient to 60% B at 3 minutes, which was held until 4 minutes followed by a linear gradient to 75% B at 5 minutes with a total run time of 6 minutes. Separation was achieved with a mm 5 lm phenyl column (Hypersil Gold; Thermo Scientific, MA, USA) maintained at 40 C. Methadone (Cerilliant, TX, USA), m/z fi 265.5, was quantified with fentanyl (Cerilliant), m/z fi 105.3, as the internal standard with the plasma standard curve linear from 1 to 1000 ng ml )1. The plasma standards were made daily using untreated canine plasma and consisted of at least six different concentrations, in addition to a blank (zero). Quality control samples, at least six, were included in each run. The run was accepted if at least 5/6 plasma standards were within 15% of the actual concentration and at least 5/6 quality control samples were within 15% of the actual concentration. The internal standard solution (100 ll, containing 500 ng ml )1 fentanyl in 15% methanol) was added to 1 ml plasma and 1 ml 0.1 N sodium hydroxide. Solid phase extraction cartridges (SPE, Bond Elut C18; Varian, CA, USA) were first conditioned with methanol (1 ml), followed by deionized (di) water (1 ml). The plasma sample or standard mixture was loaded (2.1 ml), the SPE were rinsed with 5% methanol in di water (1 ml), and the drug was eluted with methanol (1 ml). The eluate was evaporated to dryness in a water bath (temperature = 40 C) under a stream of air for no more than 35 minutes and reconstituted with 200 ll of 50% methanol and centrifuged for 5 minutes at g. The injection volume was 50 ll. The inter-day accuracy of the assay was 104 ± 9% of the actual value as determined on replicates of 5 each at 1, 10, and 100 ng ml )1 and the coefficient of variation was 9%. The limit of quantification was 1ngmL )1 as defined by the lowest point on the standard curve with measured concentrations within 15% of the actual concentration. Noncompartmental pharmacokinetic analyses were performed with computer software (WinNonlin 5.2; Pharsight Corporation, CA, USA). The variables calculated included the terminal half-life (T ½ ) and the area under the curve from time 0 to the last measured concentration above the limit of quantification of the analytical assay (AUC 0 LAST ) using the linear trapezoidal rule. The maximum serum concentration (C max ) and time to maximum serum concentration (T max ) were determined directly from the data. Statistical analyses were performed with computer software (Sigma Stat 3.11; Systat Software, Inc., CA, USA). The dose and pharmacokinetic parameters were compared statistically using a paired t-test, as the data were normally distributed with uniform variance. The plasma concentrations were statistically compared from 0.25 to 6 hours, the last time methadone was quantifiable in Phase 1, using a Wilcoxon Signed Rank test as the data were not normally distributed. The significance level was p < 0.05 for the statistical analyses. Results The mean doses of methadone for Phase 1 (methadone) and Phase 2 (methadone with CYP inhibitors) were 2.1 mg kg )1 (Table 1) and were not significantly different (p = 0.538). The actual doses of ketoconazole, chloramphenicol, fluoxetine and trimethoprim are presented in Table 2. In Phase 1, methadone was well tolerated and no adverse effects were noted. In Phase 2, immediately prior to administration of methadone, all dogs appeared clinically normal suggesting the CYP inhibitors were well tolerated. In Phase 2, after methadone was administered, marked sedation was noted in two of the dogs (one male and one female) and moderate sedation in the other four dogs. The two dogs that were markedly sedated also vomited 3 5 times each, hours after methadone administration. The markedly sedated female also had bradycardia (36 beats minute )1 ) and hypothermia (93 F, 33.9 C) 8 12 hours after drug administra- 226 Ó 2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38,

4 Table 1 Pharmacokinetic parameters after oral administration of methadone HCL and methadone HCL with the cytochrome P450 (CYP) inhibitors ketoconazole, chloramphenicol, fluoxetine, and trimethoprim in healthy Greyhound dogs. The p values represent statistical comparisons between the parameters after methadone alone and methadone with CYP inhibitors Parameter Units Mean Min Median Max Methadone Dose mg kg ) T ½ Hours C max ng ml ) AUC 0 LAST hour ng ml ) T max Hours Parameter Units Mean Min Median Max p-value Methadone with CYP inhibitors Dose mg kg ) T ½ Hours C max ng ml ) AUC 0 LAST hour ng ml ) T max Hours T ½, terminal half-life; C max, the maximum serum concentration; AUC 0 LAST, the area under the curve from time 0 to the last measured concentration above the limit of quantification of the analytical assay using the linear trapezoidal rule; T max, time to maximum serum concentration. Table 2 Mean and range doses of cytochrome P450 (CYP) inhibitors administered to healthy Greyhound dogs for 3 days CYP inhibitor Dose (mg kg )1 ) Mean Minimum Maximum Ketoconazole (q 24 hours) Chloramphenicol (q 12 hours) Fluoxetine (q 24 hours) Trimethoprim (q 24 hours) tion. The dog with bradycardia maintained strong peripheral pulses, her mucous membranes were pink and her capillary refill time was 1 second. The bradycardia resolved without treatment, and the hypothermia resolved with supportive care including warming blankets. Plasma methadone concentrations were significantly greater (p < 0.05) in Phase 2 from 0.5 to at least 6 hours, the last time point methadone was quantifiable in Phase 1 (Fig. 1). The C max and AUC 0 LAST for methadone were significantly larger in Phase 2 (methadone with CYP inhibitors) when compared to Phase 1 (methadone administered alone) and are presented in Table 1. The mean C max was 5.5 ng ml )1 for Phase 1 and ng ml )1 for Phase 2 (p = 0.016). The mean AUC 0 LAST was 13.1 hour ng ml )1 for Phase 1 and hour ng ml )1 for Phase 2 (p = 0.004). Selected pharmacokinetic parameters are not included (AUC to infinity, mean residence time, clearance per fraction of the dose absorbed, volume of distribution per fraction of the dose absorbed) due to the excessive extrapolation of the terminal phase of the methadone with CYP inhibitor group (up to 77% of the AUC to infinity) as these parameters may not be accurate. Discussion Previous studies have described the pharmacokinetics of intravenous (IV) methadone in dogs (Garrett et al. 1985; Schmidt et al. 1994; KuKanich et al. 2005a; KuKanich & Borum 2008). Methadone has a short half-life of approximately 2 4 hours and a rapid clearance approximating hepatic blood flow, suggesting that it has a high hepatic extraction ratio (Garrett et al. 1985; Schmidt et al. 1994; KuKanich et al. 2005a; KuKanich & Borum 2008). Drugs with a high hepatic extraction ratio (ER) often have a low oral Ó 2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38,

5 1000 Concentration (ng ml 1 ) Phase 1 (methadone) Phase 2 (methadone with CYP inhibitors) Time (hours) Figure 1 Mean ± SD plasma concentrations of methadone after oral administration of methadone HCL (actual mean dose 2.1 mg kg )1 ) and methadone HCL (actual mean dose 2.1 mg kg )1 ) with the purported cytochrome P450 inhibitors ketoconazole, chloramphenicol, fluoxetine, and trimethoprim to healthy Greyhound dogs. Plasma concentrations were significantly different (p < 0.05) from 0.5 to 6 hours, the last time point methadone was quantifiable in Phase 1. bioavailability (F) due to first pass metabolism and can be estimated by the following equation: F = 1)ER (Riviere 1999). Therefore it is not unexpected that the oral bioavailability of methadone in dogs is low. The oral bioavailability of methadone was not determined in the current study as methadone was not administered IV. Therefore the true effects of concurrent administration of CYP inhibitors on the oral bioavailability cannot be determined. Additionally, an assumption of determining the bioavailability of drugs is that the clearance of the drug remains unchanged from IV to the extravascular route of administration, which is unlikely with the current study. Nonetheless, significant increases in plasma methadone concentrations and the AUC occurred with concurrent administration of CYP inhibitors which resulted in clinically evident opioid effects such as marked sedation, bradycardia, and hypothermia. The results of the current study, oral methadone without CYP inhibitors, are similar to that of a previous study in which the oral bioavailability of methadone could not be determined in Beagle dogs as the plasma concentrations never exceeded 20 ng ml )1 (KuKanich et al. 2005a). In the current study, plasma concentrations of methadone never exceeded 20 ng ml )1 in the Greyhounds administered oral methadone without CYP inhibitors. We made an assumption that fluoxetine, norfluoxetine, and trimethoprim are CYP inhibitors in dogs as they are in humans, but there are species specific differences in the specificity of substrates, inhibitors, and inducers of the CYP enzymes (Hay Kraus et al. 2000; Lu et al. 2005). However, administration of ketoconazole, chloramphenicol, fluoxetine, and trimethoprim resulted in significantly increased maximum plasma concentrations and area under the curve compared to methadone administered without CYP inhibitors. The most likely causes of significant increases in the C max and AUC 0 LAST are increased systemic drug absorption and decreased elimination. However, the study could not differentiate which of the CYP inhibitors were responsible for inhibiting methadone metabolism in dogs. It is also unclear if a single CYP inhibitor would cause a similar effect or if multiple CYP inhibitors are needed to significantly alter methadone pharmacokinetics in dogs. Further studies are needed examining the individual CYP inhibitors and combinations of CYP inhibitors to better describe the drug interactions that occurred. Methadone is a substrate for p-glycoprotein (p-gp) mediated efflux in humans and mice (Levran et al. 2008; Hassan et al. 2009), but it has not been determined if it is a p-gp substrate in dogs. Ketoconazole is a p-gp inhibitor in dogs (Coelho et al. 2009). Therefore, p-gp inhibition in the intestines by ketoconazole could have contributed to the 228 Ó 2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38,

6 increased methadone plasma concentrations and AUC in Phase 2. A previous study (KuKanich et al. 2005a) administered ketoconazole in combination with methadone resulting in increased plasma methadone concentrations in 1/6 dogs, but it was not determined if the increase was due to p-gp inhibition, metabolism inhibition, or another mechanism. It also was not determined why the increase was only observed in one dog in the previous study. The marked opioid effects observed in the dogs may have been enhanced by p-gp inhibition in the blood brain barrier resulting in increased methadone penetration into the brain. P-glycoprotein knockout mice have significantly higher brain concentrations of methadone compared to wild-type mice (Wang et al. 2004). Species specific differences in p-gp substrates have been described in dogs (West & Mealey 2007), therefore further studies are needed examining the effect of p-gp inhibition or mutation on the central effects of methadone in dogs. The complete results for the pharmacokinetic analysis are not presented for the current study. Although the pharmacokinetic parameters for the methadone alone group could be accurately assessed, the pharmacokinetic parameters for the methadone with CYP inhibitors would result in excessive extrapolation of the terminal portion of the curve. Therefore the AUC to infinity, mean residence time, clearance per fraction of the dose absorbed, and volume of distribution per fraction of the dose absorbed, may not be accurately determined for the methadone with CYP inhibitor group. Therefore the presented results are limited to the C max, T max, T ½ and AUC 0 LAST. The study design did not anticipate the profound decrease in drug elimination that occurred. Plasma concentrations detected at 32 hours were well above the LOQ of the assay and extending sampling would better describe the terminal portion of the curve. Further studies examining the effects of CYP inhibitors on methadone pharmacokinetics should include extended sampling to account for the terminal portion of the curve. In order for methadone to be clinically useful as an oral analgesic, plasma concentrations within the therapeutic range must be achieved in a consistent manner. In humans with acute pain, therapeutic concentrations of methadone ranged from 33 to 59 ng ml )1 (Gourlay et al. 1982, 1986). In Phase 2, the plasma concentrations of methadone were >33 ng ml )1 within 60 minutes in 6/6 dogs and remained >33 ng ml )1 for 20 hours in 6/6 dogs, and above 33 ng ml )1 for 32 hours in 4/6 dogs. However, the plasma concentrations of methadone in dogs associated with analgesia have not been described, therefore further studies are needed in dogs to describe the usefulness of methadone as an analgesic in canine patients. In conclusion, co-administration of methadone with CYP inhibitors resulted in consistent and prolonged plasma concentrations of methadone. Further studies should include determination of the CYP inhibitor(s) responsible for the increased plasma concentrations of methadone. Additionally, the pharmacokinetics of the CYP inhibitors should also be assessed, as their elimination may also be altered, potentially resulting in increased adverse drug effects of the inhibitors. Due to the potential adverse drug effects of the inhibitors and nondiscriminatory CYP inhibition, alternative CYP inhibitors and combinations should be investigated to minimize the number of drugs administered and determine whether a more clinically relevant strategy can be used to administer oral methadone to dogs. Acknowledgements The authors would like to thank Comparative Medicine Group for their assistance with the study. The authors would also like to thank Kansas State University, the College of Veterinary Medicine at Kansas State University, the Department of Anatomy and Physiology at Kansas State University, the Analytical Pharmacology Laboratory at Kansas State University, and the Developing Scholars Program at Kansas State University for their support of the study. References Codd EE, Shank RP, Schupsky JJ et al. (1995) Serotonin and norepinephrine uptake inhibiting activity of centrally acting analgesics: structural determinants and role in antinociception. J Pharmacol Exp Ther 274, Coelho JC, Tucker R, Mattoon J et al. (2009) Biliary excretion of technetium-99m-sestamibi in wild-type dogs and in dogs with intrinsic (ABCB1-1Delta mutation) and extrinsic (ketoconazole treated) P-glycoprotein deficiency. J Vet Pharmacol Ther 32, Crewe HK, Lennard MS, Tucker GT et al. (1992) The effect of selective serotonin re-uptake inhibitors on cytochrome P4502D6 (CYP2D6) activity in human liver microsomes. Br J Clin Pharmacol 34, Ó 2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38,

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