- Email: [email protected]
The effect of fluconazole on oral methadone in dogs
Accepted Manuscript The effect of fluconazole on oral methadone in dogs Butch KuKanich, Kate KuKanich, David Rankin, Charles W. Locuson PII:
S1467-2987(19)30042-X
DOI:
https://doi.org/10.1016/j.vaa.2019.02.003
Reference:
VAA 363
To appear in:
Veterinary Anaesthesia and Analgesia
Received Date: 16 November 2018 Revised Date:
7 February 2019
Accepted Date: 8 February 2019
Please cite this article as: KuKanich B, KuKanich K, Rankin D, Locuson CW, The effect of fluconazole on oral methadone in dogs, Veterinary Anaesthesia and Analgesia, https://doi.org/10.1016/ j.vaa.2019.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
RESEARCH PAPER Running head (Authors) B KuKanich et al.
The effect of fluconazole on oral methadone in dogs
RI PT
Running head (short title) Effect of fluconazole on methadone in dogs
Butch KuKanicha, Kate KuKanichb, David Rankinb & Charles W Locusonc a
Department of Anatomy and Physiology and the Institute of Computational Comparative
SC
Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA b
Manhattan, KS, USA c
M AN U
Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University,
Vanderbilt University Center for Neuroscience Drug Discovery, Cool Springs Life Science
Center, Nashville, TN, USA
TE D
Correspondence: B KuKanich, Department of Anatomy and Physiology and the Institute of Computational Comparative Medicine, College of Veterinary Medicine, Kansas State University,
AC C
Abstract
EP
1800 Denison Avenue, Mosier P200, Manhattan, KS 66506, USA. E-mail: [email protected]
Objective To determine the effects of fluconazole on oral methadone pharmacokinetics and central mediated opioid effects in dogs. Study design Prospective, incomplete block. Animals A total of 12 healthy Beagle dogs.
ACCEPTED MANUSCRIPT
Methods Dogs were randomly allocated into two groups of six dogs. Four treatments (two treatments/group) were administered including: oral methadone (1 mg kg−1); oral fluconazole (5 mg kg−1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg−1); oral fluconazole
RI PT
(2.5 mg kg−1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg−1); and oral fluconazole (5 mg kg−1) every 24 hours starting 12 hours prior to oral methadone (1 mg kg−1). At least 28 days were implemented as a washout period between fluconazole treatments. Rectal
SC
temperature (RT), heart rate (HR), respiratory rate (fR), sedation scores and blood samples were obtained for 24 hours after methadone administration. Plasma drug concentrations were
M AN U
measured with liquid chromatography/mass spectrometry.
Results Significantly higher maximum plasma methadone concentration (mean 25–46 ng mL−1) occurred in all treatments administered fluconazole compared with methadone alone (1.5 ng mL−1). The mean 12 hour methadone plasma concentration in the fluconazole treatments were
TE D
11–20 ng mL−1. Significantly decreased RT and variable sedation occurred in all fluconazole treatments, but no changes occurred with methadone alone. There were no differences in HR or fR among treatments.
EP
Conclusions and clinical relevance Fluconazole significantly increases the extent and duration
AC C
of oral methadone exposure in dogs resulting in significant central opioid effects.
Keywords dogs, effect, fluconazole, methadone, pharmacodynamics, pharmacokinetics.
ACCEPTED MANUSCRIPT
Introduction There are currently limited options for managing moderate to severe pain in dogs (KuKanich 2013). Opioids are safe drugs in dogs and the mainstay analgesic, but have low oral
RI PT
bioavailability, are short acting and must be administered parenterally. Non-steroidal anti-
inflammatory drugs are variably effective for moderate pain, are considered poorly effective for severe pain and can have severe gastrointestinal, renal and hepatic adverse effects that can even
SC
result in death. α2-adrenergic agonists provide effective analgesia for mild to severe pain, but are short acting, need to be administered parenterally and produce severe cardiovascular adverse
M AN U
effects even at appropriate dosages (Carter et al. 2010). A safe and effective analgesic for moderate to severe pain in dogs that can be administered orally with a reasonable dosing interval is needed.
Methadone is a safe and effective parenteral opioid approved in some countries for
TE D
perioperative pain management in dogs. At clinically relevant doses, methadone has minimal adverse cardiovascular or respiratory effects (Stanley et al. 1980; Garofalo et al. 2012; Amengual et al. 2017; Vieira et al. 2017). Although bradycardia can occur, there are minimal effects on
EP
cardiac output and blood pressure at clinically relevant doses. The reported intravenous (IV) lethal dose of methadone hydrochloride in 50% of dogs (LD50) is 29 mg kg−1, equivalent to 29–
AC C
58 times the recommended dose for clinical analgesia (Kasé et al. 1959). Previous studies have documented the rapid clearance, short half-life and poor oral
bioavailability of methadone in dogs (KuKanich et al. 2005; KuKanich & Borum 2008). Subsequent studies identified the low oral bioavailability as the result of first pass metabolism, and inhibition of cytochrome P450 (CYP) metabolism resulted in markedly increased oral bioavailability and half-life (Kukanich et al. 2011; KuKanich & KuKanich 2015). Specifically,
ACCEPTED MANUSCRIPT
chloramphenicol which is an inhibitor of CYP2B11 in dogs resulted in profound differences in the pharmacokinetics of oral methadone. Combining chloramphenicol with methadone produced effective drug concentrations that persisted for up to 48 hours depending on the dose of
RI PT
methadone. However chloramphenicol is not the best choice of an inhibitor since it has the
potential for selecting resistant bacteria in dogs and has potential adverse effects in humans including bone marrow suppression that may be experienced by clients handling
SC
chloramphenicol (Yunis 1988). Fluconazole is another drug that appears to have similar CYP inhibition effects as chloramphenicol in dogs, but fluconazole is not associated with bacterial
M AN U
resistance and bone marrow suppression. A recent study used a similar strategy of administering oral fluconazole to inhibit the metabolism of orally administered tramadol in dogs (PerezJimenez et al. 2019)
The strategy of using CYP inhibitors to enhance the oral bioavailability and duration of a
TE D
drug have resulted in several drug combinations approved for use in humans by the United States Food and Drug Administration (FDA). Examples of these combinations include Nuedexta (Avenir Pharmaceuticals Inc., CA, USA), which includes quinidine as a CYP2D inhibitor to
EP
increase the extent and duration of dextromethorphan exposure (NDA 021879) and Prezcobix (Janssen Products LP, NJ, USA) with the antiviral darunavir combined with the CYP3A inhibitor
AC C
cobicistat (NDA 205395). The CYP inhibitor used to increase the duration and extent of exposure of another drug is termed a pharmacokinetic enhancer. The purpose of this study was to assess fluconazole as a CYP inhibitor / pharmacokinetic
enhancer for oral methadone in dogs. The hypothesis was that fluconazole would enhance the oral bioavailability and duration of methadone exposure in dogs. The specific aims were to determine a dose range of fluconazole that would increase the oral bioavailability and duration of
ACCEPTED MANUSCRIPT
methadone in dogs and to document the clinical effects of oral methadone in dogs when combined with fluconazole.
RI PT
Materials and methods
The study was approved by the Institutional Animal Care and Use Committee at Kansas State University. A total of 12 purpose-bred (Marshall Bioresources, NY, USA) Beagle dogs (eight
SC
males, four females) were enrolled in the study and determined as healthy based on history, physical examination, complete blood count and serum chemistry analysis. The dogs’ age range
M AN U
was 3–4 years and body weights were 9.2–13.5 kg. The dogs were returned to the university animal housing when the study was completed.
The study consisted of an incomplete block design with six dogs per treatment. The number of dogs per treatment was chosen based on previous studies (KuKanich et al. 2011;
TE D
KuKanich & KuKanich 2015). Dogs were initially blocked by sex, then randomly assigned to two treatments based on random selection by drawing names to ensure equal distribution of sexes among treatments. Each dog was administered two treatments with ≥ 4 weeks between
EP
treatments that included fluconazole. The treatments administered were 1) oral methadone (1 mg kg−1); 2) oral fluconazole (5.0 mg kg−1) every 12 hours starting 24 hours prior to oral methadone
AC C
(1 mg kg−1); 3) oral fluconazole (2.5 mg kg−1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg−1); and 4) oral fluconazole (5.0 mg kg−1) every 24 hours starting 12 hours prior to oral methadone (1 mg kg−1). Methadone was administered to the nearest ½ of the scored 5 mg (Mallinkrodt Pharmaceuticals, MO, USA) or 10 mg tablets (Elite Pharmaceuticals Inc., NJ, US) to achieve the targeted dose. Fluconazole was administered as whole FDA approved tablets
ACCEPTED MANUSCRIPT
(50 mg; manufactured for BluePoint Laboratories by Glenmark Pharmaceuticals Ltd, India) or FDA approved suspension (25 mg dose, 10 mg mL−1; Citron Pharma, NJ, USA). In treatments 2 and 3, a total of 3 fluconazole doses (5.0 mg kg−1 or 2.5 mg kg−1,
RI PT
respectively) were administered orally every 12 hours starting 24 hours and finishing 1 hour prior to a single methadone dose. In treatment 4, 2 doses of fluconazole (5.0 mg kg−1) were
administered orally, with the first 12 hours prior to a single methadone dose and the second 12
SC
hours after methadone administration. Blood was obtained using a preplaced jugular vein catheter (19 gauge, 20.3 cm; Argon Medical Devices Inc., TX, USA) prior to methadone
M AN U
administration and at 20 and 40 minutes and 1, 2, 4, 6, 8, 12 and 24 hours after methadone administration. Plasma was separated and stored frozen at −70 °C prior to drug analysis by liquid chromatography with mass spectrometry.
Immediately prior to each hourly blood collection, heart rate (HR) was obtained by
TE D
thoracic auscultation, respiratory rate (fR) was obtained by observation of thoracic excursions and rectal temperature (RT) using the same thermometer (ReliOn, Model 144-602-000; MABIS Healthcare Inc., IL, US) for each reading. Sedation was also assessed using a categorical scale:
EP
none, no apparent sedation; mild, drowsy but still active; moderate, drowsy with glazed eyes, reluctant but able to walk without assistance; heavy, very drowsy and unable to walk or requires
AC C
assistance to walk.
Plasma was analyzed for methadone (m/z 310 > 265) and fluconazole (m/z 307 > 220)
concentrations by ultra-high pressure liquid chromatography (UPLC) (Acquity Prominence UPLC; Waters Corp., MA, USA) with triple quadrupole mass spectrometry (TQD; Waters Corp.) using methadone d9 (m/z 319 > 268) and voriconazole (m/z 350 > 281) as the internal standards. Methadone plasma concentrations were determined after plasma extraction using pass
ACCEPTED MANUSCRIPT
through sample preparation plates (Ostro Pass-through Sample Preparation Plate; Waters Corp.). Plasma 50 µL was added to 50 µL of acetonitrile with 0.1% formic acid, vortexed, then added to the well of the separation plate which contained 100 µL of acetonitrile with 0.1% formic acid and
RI PT
20 ng mL−1 methadone d9. The pass through sample preparation plates were vortexed and then positive pressure was applied using nitrogen gas. The collected fluid was then directly injected with a 2 µL injection volume. The mobile phase consisted of deionized water with 0.1% formic
SC
acid (A) and acetonitrile with 0.1% formic acid (B) using the following gradient: time 0, 85% A followed by a linear gradient to 5% A at 0.8 minutes which was held until 1.2 minutes and then a
M AN U
linear gradient to 85% A with a total run time of 2 minutes with separation achieved using a column (Acquity UPLC column, HSS T3, 1.8 u, 2.1 × 50 mm; Waters Corp.) maintained at 40 °C using a flow rate of 0.6 mL minute−1. The analytical limit of quantification was 0.5 ng mL−1. The accuracy of the assay was 114.1, 105.4 and 112.5% at 1, 10 and 100 ng mL−1 while the
TE D
precision was 14.9, 5.6 and 9.0% at 1, 10 and 100 ng mL−1.
Fluconazole plasma concentrations were determined after plasma extraction using pass through sample preparation plates (Ostro Pass-through Sample Preparation Plate; Waters Corp.).
EP
Plasma 15 µL was added to 100 µL acetonitrile with 0.1% formic acid, containing 1 µg mL−1 voriconazole to the well of the separation plate, vortexed and then positive pressure was applied
AC C
using nitrogen gas. Deionized water, 110 µL was added to the collected fluid, vortexed and 2 µL injected. The mobile phase consisted of deionized water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) using the following gradient: time 0, 85% A followed by a linear gradient to 5% A at 0.8 minutes which was held until 1.2 minutes and then a linear gradient to 85% A with a total run time of 2 minutes with separation achieved using a column (Acquity UPLC column, HSS T3, 1.8 u, 2.1 × 50 mm; Waters Corp.) maintained at 40 °C using a
ACCEPTED MANUSCRIPT
flow rate of 0.6 mL minute−1. The analytical limit of quantification was 0.2 µg mL−1. The accuracy of the assay was 92.6, 97.5 and 90.4% at 0.5, 10 and 50 µg mL−1 while the precision was 8.7, 8.0 and 6.2% at 0.5, 10 and 50 µg mL−1.
RI PT
Pharmacokinetic analyses were performed with Phoenix 64 (Certara USA Inc., NJ, USA) using noncompartmental methods. The maximum plasma concentration (Cmax) and time to Cmax (tmax) were determined directly from the data. The terminal rate constant (λz) was determined by
SC
log-linear regression of the time points on the terminal portion of the curve and the terminal halflife (t½) was calculated using the formula (0.693 • λz−1). The areas under the curve (AUC) were
M AN U
calculated using the linear trapezoidal method. The AUC from time 0–24 hours (AUC0–24) was determined with the measured AUC above the analytical lower limit of quantification from time 0 to 24 hours and the AUC extrapolated to infinity (AUC∞) was calculated by adding the AUC0– 24 + (concentration at 24 hours • λz−1). The relative fraction of the dose absorbed (Frel) was
TE D
calculated by individually dividing the geometric mean AUC0–24 for the fluconazole treatments
Statistical analysis
EP
(dose normalized) by the AUC0–24 of the methadone alone treatment (also dose normalized).
Statistical analyses of pharmacokinetic and pharmacodynamic data (HR, RT, fR) were performed
AC C
with SigmaPlot 12.5 (Systat Software Inc., CA, USA). Pharmacokinetic parameters were assessed for differences using the Kruskal–Wallis one way analysis of variance (ANOVA) on Ranks with the Student–Newman–Keuls method for all pairwise multiple comparisons. Assessment of pharmacodynamic parameters for differences used an ANOVA with the Holm– Sidak method for multiple comparisons versus control (methadone alone treatment). Statistical significance was set to p < 0.05. Moderate and heavy sedation scores are reported based on the
ACCEPTED MANUSCRIPT
total number of observations after methadone administration per dog (7 time points × six dogs
Results
RI PT
per treatment = 42 observations per treatment).
The actual mean (range) doses of fluconazole in the 2.5 and 5.0 mg kg−1 treatments were 2.3 (1.9–2.4) mg kg−1 and 4.6 (3.7–5.4) mg kg−1, respectively. The mean (range) doses of methadone
SC
hydrochloride were 0.95 (0.85–1.08) mg kg−1 across the treatments.
The low and sporadic measurable plasma methadone concentrations after methadone
M AN U
alone precluded calculation of some pharmacokinetic parameters such as t½ (Fig. 1). The methadone AUC and the Cmax for the treatments containing fluconazole were significantly higher than the methadone alone treatment (p < 0.05; Table 1). There was no significant difference between the methadone AUC or Cmax among the treatments administered fluconazole. The Frel of
TE D
methadone with fluconazole, compared with methadone alone were 9,805 to 17,594% (98–176fold increases). The mean t½ of methadone when administered with fluconazole was 8.99–9.49 hours.
EP
The plasma concentrations of fluconazole were proportional to the total daily dose. The geometric mean (range) peak concentrations after fluconazole (2.5 mg kg−1) administered orally
AC C
every 12 hours was 6.95 (6.16–7.78) µg mL−1. The peak concentrations after 5.0 mg kg−1 orally every 24 hours, geometric mean (range), was 6.45 (5.06–8.43) µg mL−1. The peak concentrations after fluconazole (5.0 mg kg−1) administered orally every 12 hours, geometric mean (range), was 13.84 (8.47–17.75) µg mL−1 (Fig. 2). Significant effects on RT were noted among the treatments (p < 0.05; Fig. 3). All treatments that included fluconazole had significantly decreased RT at 4, 6 and 8 hours after
ACCEPTED MANUSCRIPT
methadone administration compared with methadone alone. In addition, in the treatment where fluconazole (2.5 mg kg−1) was administered every 12 hours, the RT was significantly lower at 2 hours compared with methadone alone, but not with the other treatments including fluconazole.
RI PT
The HR was decreased in the treatments administered fluconazole and methadone (Fig. 4). Statistical analysis of HR was not performed because of the high variability and low post hoc power (0.049), which was much lower than targeted power (0.8). The lowest measured HR in
SC
any dog was 52 beats minute−1 at 2 hours in one dog administered methadone with fluconazole (2.5 mg kg−1) every 12 hours and in one dog at 1 hour administered methadone with fluconazole
M AN U
(5 mg kg−1) every 24 hours. The fR was very variable throughout the study.
Sedation was not observed in the methadone alone treatment (Table 2). The methadone with fluconazole (2.5 mg kg−1) every 12 hours treatment had dogs with moderate sedation at 3/42 time points and heavy sedation at 0/42 time points. The methadone with fluconazole (5 mg kg−1)
TE D
every 12 hours treatment had dogs with moderate sedation at 6/42 time points and heavy sedation at 2/42 time points. The methadone with fluconazole (5 mg kg−1) every 24 hours treatment had dogs with moderate sedation at 8/42 time points and heavy sedation at 1/42 time
EP
points. All of the heavy sedation time points occurred in the same two dogs. All of the dogs were
AC C
capable of walking despite variable degrees of sedation.
Discussion
This study demonstrated the ability of fluconazole when administered at a variety of oral dosages (2.5 mg kg−1 every 12 hours, 5.0 mg kg−1 every 12 hours and 5.0 mg kg−1 every 24 hours) to enhance the oral bioavailability of methadone and its duration of exposure. Therefore fluconazole is a pharmacokinetics enhancer of methadone. Significant increases in total drug
ACCEPTED MANUSCRIPT
exposure, as measured by the AUC and Cmax occurred compared with oral methadone alone. These results are similar to previous reports in which chloramphenicol significantly increased oral methadone exposure and duration in dogs (KuKanich et al. 2011; KuKanich & KuKanich
RI PT
2015).
Dogs administered fluconazole (4 mg kg−1) orally for two doses 24 and 12 hour prior to tramadol administration resulted in significant increases in tramadol plasma concentrations at 4
SC
hours after tramadol administration (median 215 ng mL−1) compared with tramadol without fluconazole (median 6.4 ng mL−1) (Perez-Jimenez et al. 2019). The effects of fluconazole on the
M AN U
half-life or persistence of oral tramadol were not reported. Significant inhibition of tramadol to N-desmethyltramadol and N,O didesmethyltramadol also occurred owing to inhibition of tramadol metabolism to its inactive metabolites. The formation of the active tramadol metabolite (O-desmethyltramadol) was increased due to a different CYP responsible for that metabolic
TE D
pathway. The metabolites of methadone are inactive and have not been well described in dogs (Garret et al. 1985). As a result we did not quantify changes in methadone metabolic pathways or metabolite formation.
EP
An advantage of using fluconazole for increasing methadone exposure compared with chloramphenicol is that chloramphenicol is associated with aplastic anemia in humans. It is
AC C
unknown if casual exposure to chloramphenicol (e.g. handling tablets, a child chewing on a tablet, etc.) can result in aplastic anemia in humans (Yunis 1988). Chloramphenicol may also select for bacterial resistance. In contrast, there have not been reports of severe adverse effects of fluconazole in humans from casual exposure. Fluconazole administration does have the potential for selecting resistant fungal organisms as demonstrated in humans with chronic administration or immune suppression
ACCEPTED MANUSCRIPT
(White et al. 1998). Fungal species are normal gastrointestinal microbiota in dogs (e.g. Candida albicans) (Foster et al. 2013) and chronic administration may select for resistant organisms. We anticipate that the potential clinical use of fluconazole to prolong the action of methadone would
RI PT
be primarily short-term (e.g. following trauma and surgery) and, as such, fluconazole would be expected to have less of an impact on the microbiome. Long-term use would be more likely to alter the microbiome (e.g. osteoarthritis, cancer pain), however, further studies are needed to
SC
describe the specific effect in dogs and associated risk factors.
Since many systemic fungal infections are from environmental sources (e.g.
M AN U
histoplasmosis, blastomycosis, coccidiomycosis, aspergillosis), administration of fluconazole would be expected to have minor impacts on the susceptibility profile of those fungal diseases. Some researchers hypothesize that the widespread agricultural use of antifungal drugs may have a substantial impact on susceptibility profiles of these organisms resulting from the
al. 2017).
TE D
environmental application of similar azole antifungals (Hof 2001; Azevedo et al. 2015; Berger et
To determine the best scenario to increase methadone drug exposure, fluconazole was
EP
administered at least 12 hours prior to methadone to allow time for fluconazole to interact with drug metabolizing enzymes. A duration of less than 12 hours preadministration was not assessed
AC C
in this study, and it is not known if shorter times or if administration of a first dose concurrently with methadone would produce similar effects. Since fluconazole is a competitive/noncompetitive CYP inhibitor in humans (versus a CYP inactivator), the authors hypothesize fluconazole has a similar mechanism of CYP inhibition in dogs that would result in metabolism inhibition more rapidly than 12 hours (Nivoix et al. 2008). Further studies should
ACCEPTED MANUSCRIPT
assess the effects of shorter pretreatment times and concurrent administration of fluconazole on oral methadone drug exposure in dogs. It is interesting to note that the mean methadone AUC0-24 and Cmax nearly doubled when
RI PT
the dose (and plasma concentrations) of fluconazole were doubled (fluconazole 2.5 mg kg−1 administered orally every 12 hours to 5 mg kg−1 every 12 hours). These data suggest there may be a dose-dependent effect of fluconazole on methadone (and potentially other substrate)
SC
metabolism when administered short-term to dogs. The lack of statistical difference in the
methadone AUC0-24 and Cmax may have been a consequence of the variability of the data. Studies
M AN U
assessing a larger dose range using a larger sample size are needed to fully document the dose dependency of fluconazole on methadone pharmacokinetics.
Central opioid effects were documented in the dogs treated with methadone and fluconazole, but not in the dogs treated with methadone alone. Body temperature decreases in
TE D
dogs may be from activation of opioid receptors in the hypothalamus resulting in a decrease in the thermoregulatory set point, often causing panting (Adler et al. 1988). Decreases in body temperature by opioids occurs in parallel to analgesic effects (Vaupel & Jasinski 1997; Guedes et
EP
al. 2008) which is not surprising since both responses arise from activation of opioid receptors within the central nervous system. As such, changes in body temperature after opioid
AC C
administration is a marker for other central opioid effects, including analgesia. Moderate to heavy sedation was variably noted in dogs administered methadone with
fluconazole. In two of these dogs, sedation was heavy at 3 time points. Sedation is also a central mediated opioid effect and is often desirable in the perioperative setting to facilitate procedures such as catheter placement, and during recovery to prevent delirium and self-injury. All of the dogs could walk despite variable degrees of sedation. Although moderate to heavy sedation is
ACCEPTED MANUSCRIPT
undesirable in long-term drug administration, sedation from methadone is expected to be dosedependent such that decreasing the methadone dose could decrease the degree of sedation. This study was a single dose, proof of concept study and was not meant to be a definitive dose finding
to provide both an appropriate level of sedation and analgesia.
SC
Conclusion
RI PT
study. Further studies should assess a range of methadone doses in dogs to achieve optimal doses
Oral methadone when combined with oral fluconazole as a pharmacokinetic enhancer provided
M AN U
consistent methadone drug exposure and opioid mediated effects. Coadministration of a single methadone dose with alternate fluconazole regimens were well tolerated. Further studies should assess the effects and adverse effects from multiple doses of fluconazole with methadone. The optimal amount of time, if any, that fluconazole needs to be administered prior to methadone has
Acknowledgements
TE D
not been determined.
EP
The authors thank Jennie Kim and Daniel Comyn for their assistance with the animal phases of the study, and Dr Hyun Joo for analysis of the plasma drug concentrations, College of Veterinary
AC C
Medicine, Kansas State University. Funding was provided by the Analytical Pharmacology Laboratory at Kansas State University.
Authors’ contributions
ACCEPTED MANUSCRIPT
BK and KK: study design, data acquisition and analysis, preparation of manuscript. DR: data acquisition and analysis, preparation of manuscript. CWL: study design, preparation of
RI PT
manuscript. All authors critically reviewed the manuscript and approved the submission.
Conflict of interest statement
AC C
EP
TE D
M AN U
SC
Authors declare no conflict of interest.
ACCEPTED MANUSCRIPT
References Adler MW, Geller EB, Rosow CE, Cochin J (1988) The opioid system and temperature regulation. Annu Rev Pharmacol Toxicol 28, 429–449.
RI PT
Amengual M, Leigh H, Rioja E (2017) Postoperative respiratory effects of intravenous fentanyl compared to intravenous methadone in dogs following spinal surgery. Vet Anaesth Analg 44, 1042–1048.
SC
Azevedo MM, Faria-Ramos I, Cruz LC et al. (2015) Genesis of azole antifungal resistance from agriculture to clinical settings. J Agric Food Chem, 63, 7463–7468.
M AN U
Berger S, El Chazli Y, Babu AF, Coste AT (2017) Azole resistance in Aspergillus fumigatus: a consequence of antifungal use in agriculture? Front Microbiol 8, 1024. Carter JE, Campbell NB, Posner LP, Swanson C (2010) The hemodynamic effects of medetomidine continuous rate infusions in the dog. Vet Anaesth Analg 37, 197–206.
TE D
Foster ML, Dowd SE, Stephenson C et al. (2013) Characterization of the fungal microbiome (mycobiome) in fecal samples from dogs. Vet Med Int 2013, 658373. Garofalo NA, Teixeira Neto FJ, Pereira CD et al. (2012) Cardiorespiratory and neuroendocrine
398–404.
EP
changes induced by methadone in conscious and in isoflurane anaesthetised dogs. Vet J 194,
AC C
Garrett ER, Derendorf H, Mattha AG (1985) 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 74, 1203–1214. Guedes AG, Papich MG, Rude EP, Rider MA (2008) Pharmacokinetics and physiological effects of intravenous hydromorphone in conscious dogs. J Vet Pharmacol Ther 31, 334–343.
ACCEPTED MANUSCRIPT
Hof H (2001) Critical annotations to the use of azole antifungals for plant protection. Antimicrob Agents Chemother 45, 2987–2990. Kasé Y, Yuizono T, Yamasaki T et al. (1959) A new potent non-narcotic antitussive, 1-Methyl-
RI PT
3-di (2-thienyl) methylenepiperidine. Pharmacology and clinical efficacy. Chem Pharmaceut Bul 7, 372–377.
KuKanich B (2013) Outpatient oral analgesics in dogs and cats beyond nonsteroidal
SC
antiinflammatory drugs: an evidence-based approach. Vet Clin North Am Small Anim Pract 43, 1109–1125.
M AN U
KuKanich B, Borum SL (2008) The disposition and behavioral effects of methadone in Greyhounds. Vet Anaesth Analg 35, 242–248.
KuKanich B, KuKanich K (2015) Chloramphenicol significantly affects the pharmacokinetics of oral methadone in Greyhound dogs. Vet Anaesth Analg 42, 597–607.
TE D
Kukanich B, Kukanich KS, Rodriguez JR (2011) The effects of concurrent administration of cytochrome P-450 inhibitors on the pharmacokinetics of oral methadone in healthy dogs. Vet Anaesth Analg 38, 224–230.
EP
Kukanich B, Lascelles BD, Aman AM et al. (2005) The effects of inhibiting cytochrome P450 3A, p-glycoprotein, and gastric acid secretion on the oral bioavailability of methadone in
AC C
dogs. J Vet Pharmacol Ther 28, 461–466. Nivoix Y, Levêque D, Herbrecht R et al. (2008) The enzymatic basis of drug-drug interactions with systemic triazole antifungals. Clin Pharmacokinet 47, 779–792. Perez-Jimenez TE, Kukanich B, Joo H et al. (2019) Oral coadministration of fluconazole with tramadol markedly increases plasma and urine concentrations of tramadol and the ODesmethyltramadol metabolite in healthy dogs. Drug Metab Dispos 47, 15–25.
ACCEPTED MANUSCRIPT
Stanley TH, Liu WS, Webster LR, Johansen RK (1980) Haemodynamic effects of intravenous methadone anaesthesia in dogs. Can Anaesth Soc J 27, 52–57. Vaupel DB, Jasinski DR (1997) l-alpha-Acetylmethadol, l-alpha-acetyl-N-normethadol and l-
RI PT
alpha-acetyl-N,N-dinormethadol: comparisons with morphine and methadone in suppression of the opioid withdrawal syndrome in the dog. J Pharmacol Exp Ther 283, 833–842.
Vieira BHB, Nishimura LT, Carvalho LL et al. (2017) Cardiopulmonary and sedative effects of
SC
intravenous or epidural methadone in conscious dogs. J Vet Pharmacol Ther 40, e65–e68. White TC, Marr KA, Bowden RA (1998) Clinical, cellular, and molecular factors that contribute
M AN U
to antifungal drug resistance. Clin Microbiol Rev 11, 382–402.
Yunis AA (1988) Chloramphenicol: relation of structure to activity and toxicity. Annu Rev
AC C
EP
TE D
Pharmacol Toxicol 28, 83–100.
ACCEPTED MANUSCRIPT
Figure 1 Methadone plasma concentrations after oral methadone hydrochloride (arithmetic mean dose 0.95 mg kg−1; time 0) in six healthy Beagle dogs. Four treatments consisted of oral methadone, oral methadone administered 24 hours after oral fluconazole (5 mg kg−1) every 12
RI PT
hours for 3 doses, oral methadone administered 24 hours after oral fluconazole (2.5 mg kg−1) every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5
SC
mg kg−1) every 24 hours for 2 doses. Data are arithmetic mean ± standard deviation.
Figure 2 Fluconazole plasma concentrations after oral methadone hydrochloride (arithmetic
M AN U
mean dose 0.95 mg kg−1; time 0) in six healthy Beagle dogs. Three treatments included oral fluconazole: oral methadone administered 24 hours after oral fluconazole (5 mg kg−1) every 12 hours for 3 doses, oral methadone administered 24 hours after oral fluconazole (2.5 mg kg−1) every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5
TE D
mg kg−1) every 24 hours for 2 doses. Fluconazole was not detected in any sample from dogs administered methadone alone. Data are arithmetic mean ± standard deviation.
EP
Figure 3 Change in rectal temperature (arithmetic mean ± standard deviation) after oral administration of methadone hydrochloride (arithmetic mean dose 0.95 mg kg−1; time 0) in six
AC C
healthy Beagle dogs. Four treatments consisted of oral methadone alone, oral methadone administered 24 hours after oral fluconazole 5 mg kg−1 every 12 hours for 3 doses, oral methadone administered 24 hours after oral fluconazole 2.5 mg kg−1 every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5 mg kg−1) every 24 hours for 2 doses. * p < 0.05 for 2.5 mg kg−1 every 12 hours compared with methadone alone. † p < 0.05 for all fluconazole treatments compared with methadone alone.
ACCEPTED MANUSCRIPT
Figure 4 Change in heart rate after oral administration of methadone hydrochloride (arithmetic mean dose 0.95 mg kg−1; time 0) in six healthy Beagle dogs. Four treatments consisted of oral
RI PT
methadone alone, oral methadone administered 24 hours after oral fluconazole (5 mg kg−1) every 12 hours for 3 doses, oral methadone administered 24 hours after oral fluconazole (2.5 mg kg−1) every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5
AC C
EP
TE D
M AN U
SC
mg kg−1) every 24 hours for 2 doses.
ACCEPTED MANUSCRIPT
Table 1 Pharmacokinetics [geometric mean (range)] of a single oral dose of methadone hydrochloride (1 mg kg−1) in dogs
RI PT
administered methadone hydrochloride alone; or when administered fluconazole (2.5 mg kg−1) orally every 12 hours (total 3 doses) before methadone hydrochloride; fluconazole (5.0 mg kg−1) administered every 12 hours (total 3 doses) before methadone hydrochloride; or fluconazole (5.0 mg kg−1) administered 12 hours before and 12 hours after methadone hydrochloride. There were
Treatments Methadone
Fluconazole
Fluconazole
(2.5 mg kg−1) every 12 hours
AUC∞*
0.911
500.5 (341.6–1039.9)
358.1 (203.5–874.0)
0.002
332.1 (155.6–1286.2)
630.6 (484.3–1195.4)
442.6 (274.0–988.4)
0.104
25.4 (11.7–55.1)
45.9 (31.2–90.8)
39.6 (18.2–130.1)
0.002
na
1.5 (1.3–1.9)
TE D
270.8 (130.4–727.4)
(hour ng mL−1) Cmax
15.7 (5.7–34.6)
2.8 (1.1–5.7)
(hour ng mL−1)
(5.0 mg kg−1) every 12 (5.0 mg kg−1) every 24
17.2 (10.8–38.7)
14.8 (9.0–43.4)
EP
AUC0-24*
Fluconazole
hours
na
(%)
p - value
hours
AC C
AUCExtrap
M AN U
Parameter
SC
six dogs in each treatment and each dog was in 2 treatments.
ACCEPTED MANUSCRIPT
(ng mL−1) 0.54 (0.33–
2.24 (2–4)
2.83 (2–4)
(hour)
1.00)
t½
na
8.99 (6.63–19.97)
9.49 (7.02–18.14)
na
0.0771 (0.0347–0.1045) 0.0730 (0.0382–
M AN U
λz (1 hour−1) Frel
na
9.45 (5.88–15.53)
SC
(hour)
2.24 (1–4)
RI PT
tmax
9805
0.0988)
0.1178)
17594
12591
0.994
na
na
TE D
(%)
0.0734 (0.0446–
0.002
EP
AUCExtrap, percent of the AUC extrapolated to infinity; AUC0-24, area under the curve from time 0–24 hours; AUC∞, area under the curve extrapolated to infinity; Cmax, maximum plasma concentration; tmax, time of maximum plasma concentration; t½, terminal half-
AC C
life; Frel, relative bioavailability (expressed as %) compared with methadone alone. *Statistical comparisons were performed using dose normalized values. Significance when compared with methadone alone (p < 0.05). No significant differences occurred among treatments administered methadone with fluconazole (p < 0.05).
ACCEPTED MANUSCRIPT
Table 2 Sedation scores of dogs administered methadone (1 mg kg−1); fluconazole (2.5 mg kg−1) administered every 12 hours (total 3
RI PT
doses) before methadone; fluconazole (5.0 mg kg−1) administered every 12 hours (total 3 doses) before methadone; and fluconazole (5.0 mg kg−1) administered 12 hours before and 12 hours after methadone administration. All drugs were administered orally and the dose of methadone was the same in every treatment. Each treatment was comprised of six dogs and each dog was included in 2
SC
treatments. Shown are the numbers of dogs (n) with the designated sedation score at each time point (0–24 hours) after administration
Time
M AN U
of the indicated treatment. Treatments
(hour)
Methadone
Methadone with
Methadone with
Methadone with
fluconazole
fluconazole
mg kg−1) every 12
(5.0 mg kg−1)
(5.0 mg kg−1)
hours
every 12 hours
every 24 hours
0
0
0
0
6
0
0
0
6
0
0
0
5
1
0
0
5
1
0
0
4
1
1
0
2
3
1
0
2
2
1
1
3
1
1
1
EP
0
6
AC C
0
Heavy
6
Moderate
2
Mild
0
None
0
Heavy
0
Moderate
6
Mild
1
None
0
Heavy
Heavy
0
Moderate
Moderate
0
Mild
Mild
6
None
None
0
TE D
fluconazole (2.5
ACCEPTED MANUSCRIPT
6
0
0
0
1
3
2
0
0
3
3
0
2
1
3
0
6
6
0
0
0
0
6
0
0
0
4
1
1
2
1
3
0
8
6
0
0
0
3
3
0
0
1
4
1
0
12
6
0
0
0
6
0
0
0
1
5
0
0
24
6
0
0
0
6
0
0
0
6
0
0
0
AC C
EP
TE D
M AN U
SC
RI PT
4
3
3
0
0
2
4
0
0
6
0
0
0
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S1467-2987(19)30042-X
DOI:
https://doi.org/10.1016/j.vaa.2019.02.003
Reference:
VAA 363
To appear in:
Veterinary Anaesthesia and Analgesia
Received Date: 16 November 2018 Revised Date:
7 February 2019
Accepted Date: 8 February 2019
Please cite this article as: KuKanich B, KuKanich K, Rankin D, Locuson CW, The effect of fluconazole on oral methadone in dogs, Veterinary Anaesthesia and Analgesia, https://doi.org/10.1016/ j.vaa.2019.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
RESEARCH PAPER Running head (Authors) B KuKanich et al.
The effect of fluconazole on oral methadone in dogs
RI PT
Running head (short title) Effect of fluconazole on methadone in dogs
Butch KuKanicha, Kate KuKanichb, David Rankinb & Charles W Locusonc a
Department of Anatomy and Physiology and the Institute of Computational Comparative
SC
Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA b
Manhattan, KS, USA c
M AN U
Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University,
Vanderbilt University Center for Neuroscience Drug Discovery, Cool Springs Life Science
Center, Nashville, TN, USA
TE D
Correspondence: B KuKanich, Department of Anatomy and Physiology and the Institute of Computational Comparative Medicine, College of Veterinary Medicine, Kansas State University,
AC C
Abstract
EP
1800 Denison Avenue, Mosier P200, Manhattan, KS 66506, USA. E-mail: [email protected]
Objective To determine the effects of fluconazole on oral methadone pharmacokinetics and central mediated opioid effects in dogs. Study design Prospective, incomplete block. Animals A total of 12 healthy Beagle dogs.
ACCEPTED MANUSCRIPT
Methods Dogs were randomly allocated into two groups of six dogs. Four treatments (two treatments/group) were administered including: oral methadone (1 mg kg−1); oral fluconazole (5 mg kg−1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg−1); oral fluconazole
RI PT
(2.5 mg kg−1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg−1); and oral fluconazole (5 mg kg−1) every 24 hours starting 12 hours prior to oral methadone (1 mg kg−1). At least 28 days were implemented as a washout period between fluconazole treatments. Rectal
SC
temperature (RT), heart rate (HR), respiratory rate (fR), sedation scores and blood samples were obtained for 24 hours after methadone administration. Plasma drug concentrations were
M AN U
measured with liquid chromatography/mass spectrometry.
Results Significantly higher maximum plasma methadone concentration (mean 25–46 ng mL−1) occurred in all treatments administered fluconazole compared with methadone alone (1.5 ng mL−1). The mean 12 hour methadone plasma concentration in the fluconazole treatments were
TE D
11–20 ng mL−1. Significantly decreased RT and variable sedation occurred in all fluconazole treatments, but no changes occurred with methadone alone. There were no differences in HR or fR among treatments.
EP
Conclusions and clinical relevance Fluconazole significantly increases the extent and duration
AC C
of oral methadone exposure in dogs resulting in significant central opioid effects.
Keywords dogs, effect, fluconazole, methadone, pharmacodynamics, pharmacokinetics.
ACCEPTED MANUSCRIPT
Introduction There are currently limited options for managing moderate to severe pain in dogs (KuKanich 2013). Opioids are safe drugs in dogs and the mainstay analgesic, but have low oral
RI PT
bioavailability, are short acting and must be administered parenterally. Non-steroidal anti-
inflammatory drugs are variably effective for moderate pain, are considered poorly effective for severe pain and can have severe gastrointestinal, renal and hepatic adverse effects that can even
SC
result in death. α2-adrenergic agonists provide effective analgesia for mild to severe pain, but are short acting, need to be administered parenterally and produce severe cardiovascular adverse
M AN U
effects even at appropriate dosages (Carter et al. 2010). A safe and effective analgesic for moderate to severe pain in dogs that can be administered orally with a reasonable dosing interval is needed.
Methadone is a safe and effective parenteral opioid approved in some countries for
TE D
perioperative pain management in dogs. At clinically relevant doses, methadone has minimal adverse cardiovascular or respiratory effects (Stanley et al. 1980; Garofalo et al. 2012; Amengual et al. 2017; Vieira et al. 2017). Although bradycardia can occur, there are minimal effects on
EP
cardiac output and blood pressure at clinically relevant doses. The reported intravenous (IV) lethal dose of methadone hydrochloride in 50% of dogs (LD50) is 29 mg kg−1, equivalent to 29–
AC C
58 times the recommended dose for clinical analgesia (Kasé et al. 1959). Previous studies have documented the rapid clearance, short half-life and poor oral
bioavailability of methadone in dogs (KuKanich et al. 2005; KuKanich & Borum 2008). Subsequent studies identified the low oral bioavailability as the result of first pass metabolism, and inhibition of cytochrome P450 (CYP) metabolism resulted in markedly increased oral bioavailability and half-life (Kukanich et al. 2011; KuKanich & KuKanich 2015). Specifically,
ACCEPTED MANUSCRIPT
chloramphenicol which is an inhibitor of CYP2B11 in dogs resulted in profound differences in the pharmacokinetics of oral methadone. Combining chloramphenicol with methadone produced effective drug concentrations that persisted for up to 48 hours depending on the dose of
RI PT
methadone. However chloramphenicol is not the best choice of an inhibitor since it has the
potential for selecting resistant bacteria in dogs and has potential adverse effects in humans including bone marrow suppression that may be experienced by clients handling
SC
chloramphenicol (Yunis 1988). Fluconazole is another drug that appears to have similar CYP inhibition effects as chloramphenicol in dogs, but fluconazole is not associated with bacterial
M AN U
resistance and bone marrow suppression. A recent study used a similar strategy of administering oral fluconazole to inhibit the metabolism of orally administered tramadol in dogs (PerezJimenez et al. 2019)
The strategy of using CYP inhibitors to enhance the oral bioavailability and duration of a
TE D
drug have resulted in several drug combinations approved for use in humans by the United States Food and Drug Administration (FDA). Examples of these combinations include Nuedexta (Avenir Pharmaceuticals Inc., CA, USA), which includes quinidine as a CYP2D inhibitor to
EP
increase the extent and duration of dextromethorphan exposure (NDA 021879) and Prezcobix (Janssen Products LP, NJ, USA) with the antiviral darunavir combined with the CYP3A inhibitor
AC C
cobicistat (NDA 205395). The CYP inhibitor used to increase the duration and extent of exposure of another drug is termed a pharmacokinetic enhancer. The purpose of this study was to assess fluconazole as a CYP inhibitor / pharmacokinetic
enhancer for oral methadone in dogs. The hypothesis was that fluconazole would enhance the oral bioavailability and duration of methadone exposure in dogs. The specific aims were to determine a dose range of fluconazole that would increase the oral bioavailability and duration of
ACCEPTED MANUSCRIPT
methadone in dogs and to document the clinical effects of oral methadone in dogs when combined with fluconazole.
RI PT
Materials and methods
The study was approved by the Institutional Animal Care and Use Committee at Kansas State University. A total of 12 purpose-bred (Marshall Bioresources, NY, USA) Beagle dogs (eight
SC
males, four females) were enrolled in the study and determined as healthy based on history, physical examination, complete blood count and serum chemistry analysis. The dogs’ age range
M AN U
was 3–4 years and body weights were 9.2–13.5 kg. The dogs were returned to the university animal housing when the study was completed.
The study consisted of an incomplete block design with six dogs per treatment. The number of dogs per treatment was chosen based on previous studies (KuKanich et al. 2011;
TE D
KuKanich & KuKanich 2015). Dogs were initially blocked by sex, then randomly assigned to two treatments based on random selection by drawing names to ensure equal distribution of sexes among treatments. Each dog was administered two treatments with ≥ 4 weeks between
EP
treatments that included fluconazole. The treatments administered were 1) oral methadone (1 mg kg−1); 2) oral fluconazole (5.0 mg kg−1) every 12 hours starting 24 hours prior to oral methadone
AC C
(1 mg kg−1); 3) oral fluconazole (2.5 mg kg−1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg−1); and 4) oral fluconazole (5.0 mg kg−1) every 24 hours starting 12 hours prior to oral methadone (1 mg kg−1). Methadone was administered to the nearest ½ of the scored 5 mg (Mallinkrodt Pharmaceuticals, MO, USA) or 10 mg tablets (Elite Pharmaceuticals Inc., NJ, US) to achieve the targeted dose. Fluconazole was administered as whole FDA approved tablets
ACCEPTED MANUSCRIPT
(50 mg; manufactured for BluePoint Laboratories by Glenmark Pharmaceuticals Ltd, India) or FDA approved suspension (25 mg dose, 10 mg mL−1; Citron Pharma, NJ, USA). In treatments 2 and 3, a total of 3 fluconazole doses (5.0 mg kg−1 or 2.5 mg kg−1,
RI PT
respectively) were administered orally every 12 hours starting 24 hours and finishing 1 hour prior to a single methadone dose. In treatment 4, 2 doses of fluconazole (5.0 mg kg−1) were
administered orally, with the first 12 hours prior to a single methadone dose and the second 12
SC
hours after methadone administration. Blood was obtained using a preplaced jugular vein catheter (19 gauge, 20.3 cm; Argon Medical Devices Inc., TX, USA) prior to methadone
M AN U
administration and at 20 and 40 minutes and 1, 2, 4, 6, 8, 12 and 24 hours after methadone administration. Plasma was separated and stored frozen at −70 °C prior to drug analysis by liquid chromatography with mass spectrometry.
Immediately prior to each hourly blood collection, heart rate (HR) was obtained by
TE D
thoracic auscultation, respiratory rate (fR) was obtained by observation of thoracic excursions and rectal temperature (RT) using the same thermometer (ReliOn, Model 144-602-000; MABIS Healthcare Inc., IL, US) for each reading. Sedation was also assessed using a categorical scale:
EP
none, no apparent sedation; mild, drowsy but still active; moderate, drowsy with glazed eyes, reluctant but able to walk without assistance; heavy, very drowsy and unable to walk or requires
AC C
assistance to walk.
Plasma was analyzed for methadone (m/z 310 > 265) and fluconazole (m/z 307 > 220)
concentrations by ultra-high pressure liquid chromatography (UPLC) (Acquity Prominence UPLC; Waters Corp., MA, USA) with triple quadrupole mass spectrometry (TQD; Waters Corp.) using methadone d9 (m/z 319 > 268) and voriconazole (m/z 350 > 281) as the internal standards. Methadone plasma concentrations were determined after plasma extraction using pass
ACCEPTED MANUSCRIPT
through sample preparation plates (Ostro Pass-through Sample Preparation Plate; Waters Corp.). Plasma 50 µL was added to 50 µL of acetonitrile with 0.1% formic acid, vortexed, then added to the well of the separation plate which contained 100 µL of acetonitrile with 0.1% formic acid and
RI PT
20 ng mL−1 methadone d9. The pass through sample preparation plates were vortexed and then positive pressure was applied using nitrogen gas. The collected fluid was then directly injected with a 2 µL injection volume. The mobile phase consisted of deionized water with 0.1% formic
SC
acid (A) and acetonitrile with 0.1% formic acid (B) using the following gradient: time 0, 85% A followed by a linear gradient to 5% A at 0.8 minutes which was held until 1.2 minutes and then a
M AN U
linear gradient to 85% A with a total run time of 2 minutes with separation achieved using a column (Acquity UPLC column, HSS T3, 1.8 u, 2.1 × 50 mm; Waters Corp.) maintained at 40 °C using a flow rate of 0.6 mL minute−1. The analytical limit of quantification was 0.5 ng mL−1. The accuracy of the assay was 114.1, 105.4 and 112.5% at 1, 10 and 100 ng mL−1 while the
TE D
precision was 14.9, 5.6 and 9.0% at 1, 10 and 100 ng mL−1.
Fluconazole plasma concentrations were determined after plasma extraction using pass through sample preparation plates (Ostro Pass-through Sample Preparation Plate; Waters Corp.).
EP
Plasma 15 µL was added to 100 µL acetonitrile with 0.1% formic acid, containing 1 µg mL−1 voriconazole to the well of the separation plate, vortexed and then positive pressure was applied
AC C
using nitrogen gas. Deionized water, 110 µL was added to the collected fluid, vortexed and 2 µL injected. The mobile phase consisted of deionized water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) using the following gradient: time 0, 85% A followed by a linear gradient to 5% A at 0.8 minutes which was held until 1.2 minutes and then a linear gradient to 85% A with a total run time of 2 minutes with separation achieved using a column (Acquity UPLC column, HSS T3, 1.8 u, 2.1 × 50 mm; Waters Corp.) maintained at 40 °C using a
ACCEPTED MANUSCRIPT
flow rate of 0.6 mL minute−1. The analytical limit of quantification was 0.2 µg mL−1. The accuracy of the assay was 92.6, 97.5 and 90.4% at 0.5, 10 and 50 µg mL−1 while the precision was 8.7, 8.0 and 6.2% at 0.5, 10 and 50 µg mL−1.
RI PT
Pharmacokinetic analyses were performed with Phoenix 64 (Certara USA Inc., NJ, USA) using noncompartmental methods. The maximum plasma concentration (Cmax) and time to Cmax (tmax) were determined directly from the data. The terminal rate constant (λz) was determined by
SC
log-linear regression of the time points on the terminal portion of the curve and the terminal halflife (t½) was calculated using the formula (0.693 • λz−1). The areas under the curve (AUC) were
M AN U
calculated using the linear trapezoidal method. The AUC from time 0–24 hours (AUC0–24) was determined with the measured AUC above the analytical lower limit of quantification from time 0 to 24 hours and the AUC extrapolated to infinity (AUC∞) was calculated by adding the AUC0– 24 + (concentration at 24 hours • λz−1). The relative fraction of the dose absorbed (Frel) was
TE D
calculated by individually dividing the geometric mean AUC0–24 for the fluconazole treatments
Statistical analysis
EP
(dose normalized) by the AUC0–24 of the methadone alone treatment (also dose normalized).
Statistical analyses of pharmacokinetic and pharmacodynamic data (HR, RT, fR) were performed
AC C
with SigmaPlot 12.5 (Systat Software Inc., CA, USA). Pharmacokinetic parameters were assessed for differences using the Kruskal–Wallis one way analysis of variance (ANOVA) on Ranks with the Student–Newman–Keuls method for all pairwise multiple comparisons. Assessment of pharmacodynamic parameters for differences used an ANOVA with the Holm– Sidak method for multiple comparisons versus control (methadone alone treatment). Statistical significance was set to p < 0.05. Moderate and heavy sedation scores are reported based on the
ACCEPTED MANUSCRIPT
total number of observations after methadone administration per dog (7 time points × six dogs
Results
RI PT
per treatment = 42 observations per treatment).
The actual mean (range) doses of fluconazole in the 2.5 and 5.0 mg kg−1 treatments were 2.3 (1.9–2.4) mg kg−1 and 4.6 (3.7–5.4) mg kg−1, respectively. The mean (range) doses of methadone
SC
hydrochloride were 0.95 (0.85–1.08) mg kg−1 across the treatments.
The low and sporadic measurable plasma methadone concentrations after methadone
M AN U
alone precluded calculation of some pharmacokinetic parameters such as t½ (Fig. 1). The methadone AUC and the Cmax for the treatments containing fluconazole were significantly higher than the methadone alone treatment (p < 0.05; Table 1). There was no significant difference between the methadone AUC or Cmax among the treatments administered fluconazole. The Frel of
TE D
methadone with fluconazole, compared with methadone alone were 9,805 to 17,594% (98–176fold increases). The mean t½ of methadone when administered with fluconazole was 8.99–9.49 hours.
EP
The plasma concentrations of fluconazole were proportional to the total daily dose. The geometric mean (range) peak concentrations after fluconazole (2.5 mg kg−1) administered orally
AC C
every 12 hours was 6.95 (6.16–7.78) µg mL−1. The peak concentrations after 5.0 mg kg−1 orally every 24 hours, geometric mean (range), was 6.45 (5.06–8.43) µg mL−1. The peak concentrations after fluconazole (5.0 mg kg−1) administered orally every 12 hours, geometric mean (range), was 13.84 (8.47–17.75) µg mL−1 (Fig. 2). Significant effects on RT were noted among the treatments (p < 0.05; Fig. 3). All treatments that included fluconazole had significantly decreased RT at 4, 6 and 8 hours after
ACCEPTED MANUSCRIPT
methadone administration compared with methadone alone. In addition, in the treatment where fluconazole (2.5 mg kg−1) was administered every 12 hours, the RT was significantly lower at 2 hours compared with methadone alone, but not with the other treatments including fluconazole.
RI PT
The HR was decreased in the treatments administered fluconazole and methadone (Fig. 4). Statistical analysis of HR was not performed because of the high variability and low post hoc power (0.049), which was much lower than targeted power (0.8). The lowest measured HR in
SC
any dog was 52 beats minute−1 at 2 hours in one dog administered methadone with fluconazole (2.5 mg kg−1) every 12 hours and in one dog at 1 hour administered methadone with fluconazole
M AN U
(5 mg kg−1) every 24 hours. The fR was very variable throughout the study.
Sedation was not observed in the methadone alone treatment (Table 2). The methadone with fluconazole (2.5 mg kg−1) every 12 hours treatment had dogs with moderate sedation at 3/42 time points and heavy sedation at 0/42 time points. The methadone with fluconazole (5 mg kg−1)
TE D
every 12 hours treatment had dogs with moderate sedation at 6/42 time points and heavy sedation at 2/42 time points. The methadone with fluconazole (5 mg kg−1) every 24 hours treatment had dogs with moderate sedation at 8/42 time points and heavy sedation at 1/42 time
EP
points. All of the heavy sedation time points occurred in the same two dogs. All of the dogs were
AC C
capable of walking despite variable degrees of sedation.
Discussion
This study demonstrated the ability of fluconazole when administered at a variety of oral dosages (2.5 mg kg−1 every 12 hours, 5.0 mg kg−1 every 12 hours and 5.0 mg kg−1 every 24 hours) to enhance the oral bioavailability of methadone and its duration of exposure. Therefore fluconazole is a pharmacokinetics enhancer of methadone. Significant increases in total drug
ACCEPTED MANUSCRIPT
exposure, as measured by the AUC and Cmax occurred compared with oral methadone alone. These results are similar to previous reports in which chloramphenicol significantly increased oral methadone exposure and duration in dogs (KuKanich et al. 2011; KuKanich & KuKanich
RI PT
2015).
Dogs administered fluconazole (4 mg kg−1) orally for two doses 24 and 12 hour prior to tramadol administration resulted in significant increases in tramadol plasma concentrations at 4
SC
hours after tramadol administration (median 215 ng mL−1) compared with tramadol without fluconazole (median 6.4 ng mL−1) (Perez-Jimenez et al. 2019). The effects of fluconazole on the
M AN U
half-life or persistence of oral tramadol were not reported. Significant inhibition of tramadol to N-desmethyltramadol and N,O didesmethyltramadol also occurred owing to inhibition of tramadol metabolism to its inactive metabolites. The formation of the active tramadol metabolite (O-desmethyltramadol) was increased due to a different CYP responsible for that metabolic
TE D
pathway. The metabolites of methadone are inactive and have not been well described in dogs (Garret et al. 1985). As a result we did not quantify changes in methadone metabolic pathways or metabolite formation.
EP
An advantage of using fluconazole for increasing methadone exposure compared with chloramphenicol is that chloramphenicol is associated with aplastic anemia in humans. It is
AC C
unknown if casual exposure to chloramphenicol (e.g. handling tablets, a child chewing on a tablet, etc.) can result in aplastic anemia in humans (Yunis 1988). Chloramphenicol may also select for bacterial resistance. In contrast, there have not been reports of severe adverse effects of fluconazole in humans from casual exposure. Fluconazole administration does have the potential for selecting resistant fungal organisms as demonstrated in humans with chronic administration or immune suppression
ACCEPTED MANUSCRIPT
(White et al. 1998). Fungal species are normal gastrointestinal microbiota in dogs (e.g. Candida albicans) (Foster et al. 2013) and chronic administration may select for resistant organisms. We anticipate that the potential clinical use of fluconazole to prolong the action of methadone would
RI PT
be primarily short-term (e.g. following trauma and surgery) and, as such, fluconazole would be expected to have less of an impact on the microbiome. Long-term use would be more likely to alter the microbiome (e.g. osteoarthritis, cancer pain), however, further studies are needed to
SC
describe the specific effect in dogs and associated risk factors.
Since many systemic fungal infections are from environmental sources (e.g.
M AN U
histoplasmosis, blastomycosis, coccidiomycosis, aspergillosis), administration of fluconazole would be expected to have minor impacts on the susceptibility profile of those fungal diseases. Some researchers hypothesize that the widespread agricultural use of antifungal drugs may have a substantial impact on susceptibility profiles of these organisms resulting from the
al. 2017).
TE D
environmental application of similar azole antifungals (Hof 2001; Azevedo et al. 2015; Berger et
To determine the best scenario to increase methadone drug exposure, fluconazole was
EP
administered at least 12 hours prior to methadone to allow time for fluconazole to interact with drug metabolizing enzymes. A duration of less than 12 hours preadministration was not assessed
AC C
in this study, and it is not known if shorter times or if administration of a first dose concurrently with methadone would produce similar effects. Since fluconazole is a competitive/noncompetitive CYP inhibitor in humans (versus a CYP inactivator), the authors hypothesize fluconazole has a similar mechanism of CYP inhibition in dogs that would result in metabolism inhibition more rapidly than 12 hours (Nivoix et al. 2008). Further studies should
ACCEPTED MANUSCRIPT
assess the effects of shorter pretreatment times and concurrent administration of fluconazole on oral methadone drug exposure in dogs. It is interesting to note that the mean methadone AUC0-24 and Cmax nearly doubled when
RI PT
the dose (and plasma concentrations) of fluconazole were doubled (fluconazole 2.5 mg kg−1 administered orally every 12 hours to 5 mg kg−1 every 12 hours). These data suggest there may be a dose-dependent effect of fluconazole on methadone (and potentially other substrate)
SC
metabolism when administered short-term to dogs. The lack of statistical difference in the
methadone AUC0-24 and Cmax may have been a consequence of the variability of the data. Studies
M AN U
assessing a larger dose range using a larger sample size are needed to fully document the dose dependency of fluconazole on methadone pharmacokinetics.
Central opioid effects were documented in the dogs treated with methadone and fluconazole, but not in the dogs treated with methadone alone. Body temperature decreases in
TE D
dogs may be from activation of opioid receptors in the hypothalamus resulting in a decrease in the thermoregulatory set point, often causing panting (Adler et al. 1988). Decreases in body temperature by opioids occurs in parallel to analgesic effects (Vaupel & Jasinski 1997; Guedes et
EP
al. 2008) which is not surprising since both responses arise from activation of opioid receptors within the central nervous system. As such, changes in body temperature after opioid
AC C
administration is a marker for other central opioid effects, including analgesia. Moderate to heavy sedation was variably noted in dogs administered methadone with
fluconazole. In two of these dogs, sedation was heavy at 3 time points. Sedation is also a central mediated opioid effect and is often desirable in the perioperative setting to facilitate procedures such as catheter placement, and during recovery to prevent delirium and self-injury. All of the dogs could walk despite variable degrees of sedation. Although moderate to heavy sedation is
ACCEPTED MANUSCRIPT
undesirable in long-term drug administration, sedation from methadone is expected to be dosedependent such that decreasing the methadone dose could decrease the degree of sedation. This study was a single dose, proof of concept study and was not meant to be a definitive dose finding
to provide both an appropriate level of sedation and analgesia.
SC
Conclusion
RI PT
study. Further studies should assess a range of methadone doses in dogs to achieve optimal doses
Oral methadone when combined with oral fluconazole as a pharmacokinetic enhancer provided
M AN U
consistent methadone drug exposure and opioid mediated effects. Coadministration of a single methadone dose with alternate fluconazole regimens were well tolerated. Further studies should assess the effects and adverse effects from multiple doses of fluconazole with methadone. The optimal amount of time, if any, that fluconazole needs to be administered prior to methadone has
Acknowledgements
TE D
not been determined.
EP
The authors thank Jennie Kim and Daniel Comyn for their assistance with the animal phases of the study, and Dr Hyun Joo for analysis of the plasma drug concentrations, College of Veterinary
AC C
Medicine, Kansas State University. Funding was provided by the Analytical Pharmacology Laboratory at Kansas State University.
Authors’ contributions
ACCEPTED MANUSCRIPT
BK and KK: study design, data acquisition and analysis, preparation of manuscript. DR: data acquisition and analysis, preparation of manuscript. CWL: study design, preparation of
RI PT
manuscript. All authors critically reviewed the manuscript and approved the submission.
Conflict of interest statement
AC C
EP
TE D
M AN U
SC
Authors declare no conflict of interest.
ACCEPTED MANUSCRIPT
References Adler MW, Geller EB, Rosow CE, Cochin J (1988) The opioid system and temperature regulation. Annu Rev Pharmacol Toxicol 28, 429–449.
RI PT
Amengual M, Leigh H, Rioja E (2017) Postoperative respiratory effects of intravenous fentanyl compared to intravenous methadone in dogs following spinal surgery. Vet Anaesth Analg 44, 1042–1048.
SC
Azevedo MM, Faria-Ramos I, Cruz LC et al. (2015) Genesis of azole antifungal resistance from agriculture to clinical settings. J Agric Food Chem, 63, 7463–7468.
M AN U
Berger S, El Chazli Y, Babu AF, Coste AT (2017) Azole resistance in Aspergillus fumigatus: a consequence of antifungal use in agriculture? Front Microbiol 8, 1024. Carter JE, Campbell NB, Posner LP, Swanson C (2010) The hemodynamic effects of medetomidine continuous rate infusions in the dog. Vet Anaesth Analg 37, 197–206.
TE D
Foster ML, Dowd SE, Stephenson C et al. (2013) Characterization of the fungal microbiome (mycobiome) in fecal samples from dogs. Vet Med Int 2013, 658373. Garofalo NA, Teixeira Neto FJ, Pereira CD et al. (2012) Cardiorespiratory and neuroendocrine
398–404.
EP
changes induced by methadone in conscious and in isoflurane anaesthetised dogs. Vet J 194,
AC C
Garrett ER, Derendorf H, Mattha AG (1985) 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 74, 1203–1214. Guedes AG, Papich MG, Rude EP, Rider MA (2008) Pharmacokinetics and physiological effects of intravenous hydromorphone in conscious dogs. J Vet Pharmacol Ther 31, 334–343.
ACCEPTED MANUSCRIPT
Hof H (2001) Critical annotations to the use of azole antifungals for plant protection. Antimicrob Agents Chemother 45, 2987–2990. Kasé Y, Yuizono T, Yamasaki T et al. (1959) A new potent non-narcotic antitussive, 1-Methyl-
RI PT
3-di (2-thienyl) methylenepiperidine. Pharmacology and clinical efficacy. Chem Pharmaceut Bul 7, 372–377.
KuKanich B (2013) Outpatient oral analgesics in dogs and cats beyond nonsteroidal
SC
antiinflammatory drugs: an evidence-based approach. Vet Clin North Am Small Anim Pract 43, 1109–1125.
M AN U
KuKanich B, Borum SL (2008) The disposition and behavioral effects of methadone in Greyhounds. Vet Anaesth Analg 35, 242–248.
KuKanich B, KuKanich K (2015) Chloramphenicol significantly affects the pharmacokinetics of oral methadone in Greyhound dogs. Vet Anaesth Analg 42, 597–607.
TE D
Kukanich B, Kukanich KS, Rodriguez JR (2011) The effects of concurrent administration of cytochrome P-450 inhibitors on the pharmacokinetics of oral methadone in healthy dogs. Vet Anaesth Analg 38, 224–230.
EP
Kukanich B, Lascelles BD, Aman AM et al. (2005) The effects of inhibiting cytochrome P450 3A, p-glycoprotein, and gastric acid secretion on the oral bioavailability of methadone in
AC C
dogs. J Vet Pharmacol Ther 28, 461–466. Nivoix Y, Levêque D, Herbrecht R et al. (2008) The enzymatic basis of drug-drug interactions with systemic triazole antifungals. Clin Pharmacokinet 47, 779–792. Perez-Jimenez TE, Kukanich B, Joo H et al. (2019) Oral coadministration of fluconazole with tramadol markedly increases plasma and urine concentrations of tramadol and the ODesmethyltramadol metabolite in healthy dogs. Drug Metab Dispos 47, 15–25.
ACCEPTED MANUSCRIPT
Stanley TH, Liu WS, Webster LR, Johansen RK (1980) Haemodynamic effects of intravenous methadone anaesthesia in dogs. Can Anaesth Soc J 27, 52–57. Vaupel DB, Jasinski DR (1997) l-alpha-Acetylmethadol, l-alpha-acetyl-N-normethadol and l-
RI PT
alpha-acetyl-N,N-dinormethadol: comparisons with morphine and methadone in suppression of the opioid withdrawal syndrome in the dog. J Pharmacol Exp Ther 283, 833–842.
Vieira BHB, Nishimura LT, Carvalho LL et al. (2017) Cardiopulmonary and sedative effects of
SC
intravenous or epidural methadone in conscious dogs. J Vet Pharmacol Ther 40, e65–e68. White TC, Marr KA, Bowden RA (1998) Clinical, cellular, and molecular factors that contribute
M AN U
to antifungal drug resistance. Clin Microbiol Rev 11, 382–402.
Yunis AA (1988) Chloramphenicol: relation of structure to activity and toxicity. Annu Rev
AC C
EP
TE D
Pharmacol Toxicol 28, 83–100.
ACCEPTED MANUSCRIPT
Figure 1 Methadone plasma concentrations after oral methadone hydrochloride (arithmetic mean dose 0.95 mg kg−1; time 0) in six healthy Beagle dogs. Four treatments consisted of oral methadone, oral methadone administered 24 hours after oral fluconazole (5 mg kg−1) every 12
RI PT
hours for 3 doses, oral methadone administered 24 hours after oral fluconazole (2.5 mg kg−1) every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5
SC
mg kg−1) every 24 hours for 2 doses. Data are arithmetic mean ± standard deviation.
Figure 2 Fluconazole plasma concentrations after oral methadone hydrochloride (arithmetic
M AN U
mean dose 0.95 mg kg−1; time 0) in six healthy Beagle dogs. Three treatments included oral fluconazole: oral methadone administered 24 hours after oral fluconazole (5 mg kg−1) every 12 hours for 3 doses, oral methadone administered 24 hours after oral fluconazole (2.5 mg kg−1) every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5
TE D
mg kg−1) every 24 hours for 2 doses. Fluconazole was not detected in any sample from dogs administered methadone alone. Data are arithmetic mean ± standard deviation.
EP
Figure 3 Change in rectal temperature (arithmetic mean ± standard deviation) after oral administration of methadone hydrochloride (arithmetic mean dose 0.95 mg kg−1; time 0) in six
AC C
healthy Beagle dogs. Four treatments consisted of oral methadone alone, oral methadone administered 24 hours after oral fluconazole 5 mg kg−1 every 12 hours for 3 doses, oral methadone administered 24 hours after oral fluconazole 2.5 mg kg−1 every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5 mg kg−1) every 24 hours for 2 doses. * p < 0.05 for 2.5 mg kg−1 every 12 hours compared with methadone alone. † p < 0.05 for all fluconazole treatments compared with methadone alone.
ACCEPTED MANUSCRIPT
Figure 4 Change in heart rate after oral administration of methadone hydrochloride (arithmetic mean dose 0.95 mg kg−1; time 0) in six healthy Beagle dogs. Four treatments consisted of oral
RI PT
methadone alone, oral methadone administered 24 hours after oral fluconazole (5 mg kg−1) every 12 hours for 3 doses, oral methadone administered 24 hours after oral fluconazole (2.5 mg kg−1) every 12 hours for 3 doses and oral methadone administered 12 hours after oral fluconazole (5
AC C
EP
TE D
M AN U
SC
mg kg−1) every 24 hours for 2 doses.
ACCEPTED MANUSCRIPT
Table 1 Pharmacokinetics [geometric mean (range)] of a single oral dose of methadone hydrochloride (1 mg kg−1) in dogs
RI PT
administered methadone hydrochloride alone; or when administered fluconazole (2.5 mg kg−1) orally every 12 hours (total 3 doses) before methadone hydrochloride; fluconazole (5.0 mg kg−1) administered every 12 hours (total 3 doses) before methadone hydrochloride; or fluconazole (5.0 mg kg−1) administered 12 hours before and 12 hours after methadone hydrochloride. There were
Treatments Methadone
Fluconazole
Fluconazole
(2.5 mg kg−1) every 12 hours
AUC∞*
0.911
500.5 (341.6–1039.9)
358.1 (203.5–874.0)
0.002
332.1 (155.6–1286.2)
630.6 (484.3–1195.4)
442.6 (274.0–988.4)
0.104
25.4 (11.7–55.1)
45.9 (31.2–90.8)
39.6 (18.2–130.1)
0.002
na
1.5 (1.3–1.9)
TE D
270.8 (130.4–727.4)
(hour ng mL−1) Cmax
15.7 (5.7–34.6)
2.8 (1.1–5.7)
(hour ng mL−1)
(5.0 mg kg−1) every 12 (5.0 mg kg−1) every 24
17.2 (10.8–38.7)
14.8 (9.0–43.4)
EP
AUC0-24*
Fluconazole
hours
na
(%)
p - value
hours
AC C
AUCExtrap
M AN U
Parameter
SC
six dogs in each treatment and each dog was in 2 treatments.
ACCEPTED MANUSCRIPT
(ng mL−1) 0.54 (0.33–
2.24 (2–4)
2.83 (2–4)
(hour)
1.00)
t½
na
8.99 (6.63–19.97)
9.49 (7.02–18.14)
na
0.0771 (0.0347–0.1045) 0.0730 (0.0382–
M AN U
λz (1 hour−1) Frel
na
9.45 (5.88–15.53)
SC
(hour)
2.24 (1–4)
RI PT
tmax
9805
0.0988)
0.1178)
17594
12591
0.994
na
na
TE D
(%)
0.0734 (0.0446–
0.002
EP
AUCExtrap, percent of the AUC extrapolated to infinity; AUC0-24, area under the curve from time 0–24 hours; AUC∞, area under the curve extrapolated to infinity; Cmax, maximum plasma concentration; tmax, time of maximum plasma concentration; t½, terminal half-
AC C
life; Frel, relative bioavailability (expressed as %) compared with methadone alone. *Statistical comparisons were performed using dose normalized values. Significance when compared with methadone alone (p < 0.05). No significant differences occurred among treatments administered methadone with fluconazole (p < 0.05).
ACCEPTED MANUSCRIPT
Table 2 Sedation scores of dogs administered methadone (1 mg kg−1); fluconazole (2.5 mg kg−1) administered every 12 hours (total 3
RI PT
doses) before methadone; fluconazole (5.0 mg kg−1) administered every 12 hours (total 3 doses) before methadone; and fluconazole (5.0 mg kg−1) administered 12 hours before and 12 hours after methadone administration. All drugs were administered orally and the dose of methadone was the same in every treatment. Each treatment was comprised of six dogs and each dog was included in 2
SC
treatments. Shown are the numbers of dogs (n) with the designated sedation score at each time point (0–24 hours) after administration
Time
M AN U
of the indicated treatment. Treatments
(hour)
Methadone
Methadone with
Methadone with
Methadone with
fluconazole
fluconazole
mg kg−1) every 12
(5.0 mg kg−1)
(5.0 mg kg−1)
hours
every 12 hours
every 24 hours
0
0
0
0
6
0
0
0
6
0
0
0
5
1
0
0
5
1
0
0
4
1
1
0
2
3
1
0
2
2
1
1
3
1
1
1
EP
0
6
AC C
0
Heavy
6
Moderate
2
Mild
0
None
0
Heavy
0
Moderate
6
Mild
1
None
0
Heavy
Heavy
0
Moderate
Moderate
0
Mild
Mild
6
None
None
0
TE D
fluconazole (2.5
ACCEPTED MANUSCRIPT
6
0
0
0
1
3
2
0
0
3
3
0
2
1
3
0
6
6
0
0
0
0
6
0
0
0
4
1
1
2
1
3
0
8
6
0
0
0
3
3
0
0
1
4
1
0
12
6
0
0
0
6
0
0
0
1
5
0
0
24
6
0
0
0
6
0
0
0
6
0
0
0
AC C
EP
TE D
M AN U
SC
RI PT
4
3
3
0
0
2
4
0
0
6
0
0
0
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT