Pharmacokinetics of intravenous and oral amitriptyline and its active metabolite nortriptyline in Greyhound dogs

Veterinary Anaesthesia and Analgesia, 2015

doi:10.1111/vaa.12248

RESEARCH PAPER

Pharmacokinetics of intravenous and oral amitriptyline and its active metabolite nortriptyline in Greyhound dogs Christopher Norkus*, David Rankin* & Butch KuKanich† *Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA †Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA

Correspondence: Christopher Norkus, Veterinary Health Center, Kansas State University College of Veterinary Medicine, 1800 Denison Avenue, Manhattan, KS 66506, USA. E-mail: [email protected]

Abstract Objective To evaluate the pharmacokinetics of amitriptyline and its active metabolite nortriptyline after intravenous (IV) and oral amitriptyline administration in healthy dogs. Study design Prospective randomized experiment. Animals Five healthy Greyhound dogs (three males and two females) aged 2–4 years and weighing 32.5–39.7 kg. Methods After jugular vein catheterization, dogs were administered a single oral or IV dose of amitriptyline (4 mg kg
1). Blood samples were collected at predetermined time points from baseline (0 hours) to 32 hours after administration and plasma concentrations of amitriptyline and nortriptyline were measured by liquid chromatography triple quadrupole mass spectrometry. Non-compartmental pharmacokinetic analyses were performed. Results Orally administered amitriptyline was well tolerated, but adverse effects were noted after IV administration. The mean maximum plasma concentration (CMAX) of amitriptyline was 27.4 ng mL
1 at 1 hour and its mean terminal half-life was 4.33 hours following oral amitriptyline. Bioavailability of oral amitriptyline was 6%. The mean CMAX of nortriptyline was 14.4 ng mL
1 at 2.05 hours and its mean terminal half-life was 6.20 hours following oral amitriptyline.

Conclusions and clinical relevance Amitriptyline at 4 mg kg
1 administered orally produced low amitriptyline and nortriptyline plasma concentrations. This brings into question whether the currently recommended oral dose of amitriptyline (1– 4 mg kg
1) is appropriate in dogs. Keywords amitriptyline, canine, nortriptyline, pharmacokinetics, tricyclic antidepressant.

Introduction Antidepressants are widely used for the treatment of various chronic and neuropathic conditions in humans (Dharmshaktu et al. 2012). Amitriptyline is a tricyclic antidepressant (TCA) that is believed to provide analgesia by enhancing descending inhibitory action in the spinal cord predominantly as a serotonin–norepinephrine reuptake inhibitor, but also at other sites, including the N-methyl-D-aspartate (NMDA) receptor, l and d opioid receptors, the GABAB receptor, the adenosine A1 receptor, sodium, calcium and potassium channels, and as an antiinflammatory by decreasing prostaglandin E2 and tumor necrosis factor-a (TNF-a) production (Eschalier et al. 1981; Ogata et al. 1989; AntkiewiczMichaluk et al. 1991; Cai & McCaslin 1992; Stein 1995; Skolnick et al. 1996; Mic
o et al. 1997; Gray et al. 1998, 1999; Yaron et al. 1999; Galeotti et al. 2001; Sawnyok et al. 2001; Yokogawa et al. 2002; Sudoh et al. 2003; Ignatowski et al. 2005; Sawynok et al. 2005; McCarson et al. 2006; Dharmshaktu et al. 2012). A recent case series published in the 1

Amitriptyline pharmacokinetics in dogs C Norkus et al.

veterinary literature suggested that amitriptyline may also be effective for the treatment of neuropathic and chronic pain in dogs (Cashmore et al. 2009). However, this was a small case series which did not include positive or negative controls and therefore conclusions on effectiveness should be made with caution. In humans, amitriptyline is metabolized in part to the active metabolite nortriptyline, which, in addition to amitriptyline, appears to contribute to analgesic effects after amitriptyline administration at doses of 25–125 mg day
1 (Moore et al. 2012). In humans, steady-state plasma concentrations of amitriptyline plus nortriptyline in the range of 60–220 ng mL
1 and of nortriptyline at 60– 140 ng mL
1 are associated with clinical antidepressant effects (Vandel et al. 1978). Few data are available on the pharmacokinetics of amitriptyline and its metabolites in dogs. One study reported the plasma concentrations of amitriptyline after oral administration to dogs, but did not give intravenous (IV) data or plasma concentrations of nortriptyline (Kukes et al. 2009). The purpose of this study was to evaluate the pharmacokinetics of amitriptyline and its metabolite nortriptyline after IV and oral administration in healthy Greyhound dogs. Materials and methods Animals The study was approved by the Institutional Animal Care and Use Committee at Kansas State University College of Veterinary Medicine (protocol no. 3270). Five healthy Greyhound dogs, three male and two female, aged 2–4 years and weighing 32.5–39.7 kg were used. Study design The study consisted of a crossover design in which animals were randomized (STATA Version 10.2; StataCorp LP, TX, USA) to receive either IV amitriptyline first and oral amitriptyline second or oral amitriptyline first and IV amitriptyline second. At least 7 days were allowed between treatments. A complete physical examination, complete blood count and blood chemistry panel were performed in all dogs immediately before the start of the study. All dogs were found to be healthy with no signs of systemic disease. Tablets containing either 10 mg amitriptyline (Qualitest Pharmaceuticals, AL, USA) or 25 mg 2

amitriptyline (Sandoz, Inc., NJ, USA) were administered orally to non-fasted animals at a targeted dose of 4 mg kg
1 to the nearest whole tablet. Subjects were given a standard commercially available dry kibble dog food 2 hours prior to drug administration. Blood samples consisting of 9 mL whole blood were collected using an aseptically placed 16 gauge, polyurethane, extended-use jugular catheter (Venocath-16; Abbott Laboratories Ireland Ltd, Ireland) prior to drug administration and at 10, 20, 30 and 45 minutes and at 1, 2, 3, 4, 6, 8, 12, 24 and 32 hours after drug administration, and placed in tubes containing lithium heparin (BD Vacutainer; Becton Dickinson & Co., NJ, USA). Samples were stored on ice and plasma was immediately separated by centrifugation at 3000 g for 20 minutes and stored frozen at
70 °C until analysis. The jugular catheters were flushed with 3 mL sterile 0.9% saline after each collection to maintain patency. Amitriptyline for injection was not available as an approved drug at the time of the study; therefore, amitriptyline was compounded into sterile solution by dissolving amitriptyline hydrochloride in sterile 0.9% saline for injection to a concentration of 10 mg mL
1 and then filtering it through a 0.22 lmol L
1 filter (Fisher Scientific Co. LLC, PA, USA). Fresh amitriptyline injections were made on the day of drug administration. Amitriptyline was administered IV at 4 mg kg
1 over 5 minutes through an aseptically placed 20 gauge, 3.81 cm catheter (Terumo Medical Corp., NJ, USA) located in a cephalic vein, after which 9 mL sterile 0.9% saline was administered to flush the catheter. Blood samples (9 mL) were collected from an aseptically placed jugular catheter (Venocath-16) in the same fashion and at time intervals identical to those used after oral amitriptyline. Samples were handled and processed exactly as described for those obtained after oral amitriptyline. Subjects were observed continuously for the first 12 hours and then again at 24 and 32 hours for side effects. Liquid chromatography/mass spectrometry Amitriptyline and nortriptyline plasma concentrations were determined using liquid chromatography (LC) (Shimadzu Prominence; Shimadzu Scientific Instruments, Inc., MD, USA) with mass spectrometry (MS) (API 3000; Applied Biosystems, Inc., ON, Canada). Plasma samples were processed using a protein precipitation procedure. The internal standard solution consisted of 100 ng mL
1 of

© 2015 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia

Amitriptyline pharmacokinetics in dogs C Norkus et al. amitriptyline-d3 and 100 ng mL
1 of nortriptyline-d3 in methanol with 0.1% formic acid. Plasma, 0.05 mL, was added to a microcentrifuge tube containing 0.2 mL of the internal standard solution and vortexed. The microcentrifuge tubes were centrifuged for 5 minutes at 15,000 g, after which the supernatant was transferred to an injection vial from which 0.02 mL was injected into the LC/MS. The mobile phase consisted of A: acetonitrile and B: 0.1% formic acid at a flow rate of 0.4 mL minute
1. The mobile phase started at 100% B from 0 to 1 minute with a linear gradient to 60% B at 5.5 minutes, followed by a linear gradient to 100% B at 6 minutes with a total run time of 8 minutes. Separation was achieved with a 2.1 9 50.0 mm, 5 lmol L
1 column (XBridge C18; Waters Corp., MA, USA) maintained at 40 °C. The qualifying ion for amitriptyline had a mass/ charge ratio (m/z) of 278.26. The quantifying ion had an m/z of 105.2. For nortriptyline, the qualifying ion had an m/z of 264.34 and the quantifying ion an m/z of 105.1. For the internal standard amitriptyline-d3, the qualifying ion had an m/z of 281.24 and the quantifying ion an m/z of 105.1. For the internal standard nortriptyline-d3, the qualifying ion had an m/z of 267.34 and the quantifying ion an m/z of 105.2. The analytical range of the assays for amitriptyline was 5–1000 ng mL
1. The accuracy levels of the assays for amitriptyline were 97.6%, 105.3%, 94.0%, 98.6% and 112.7% on replicates of three for the concentrations 5, 10, 25, 100 and 1000 ng mL
1, respectively. The coefficients of variation of the assays for amitriptyline were 5.7%, 6.5%, 1.9%, 1.7% and 1.0% on replicates of three for the concentrations 5, 10, 25, 100 and 1000 ng mL
1, respectively. The analytical range of the assays for nortriptyline was 5– 250 ng mL
1. The accuracy levels of the assays for nortriptyline were 101.6%, 95.8%, 101.3% and 100.2% on replicates of three for the concentrations 5, 10, 25 and 100 ng mL
1, respectively. The coefficients of variation of the assays for nortriptyline were 5.1%, 1.0%, 1.3% and 1.7% on replicates of three for the concentrations 5, 10, 25 and 100 ng mL
1, respectively. Pharmacokinetic and data analysis Non-compartmental pharmacokinetic analyses were performed with computer software (WinNonlin Version 5.2; Pharsight Corp., CA, USA). The

pharmacokinetics calculated after IV administration included the area under the curve (AUC) from time 0 to the last time point above the analytical lower limit of quantification (LLoQ), the AUC extrapolated to infinity (AUCINF), the percentage of the AUC extrapolated to infinity (AUC % extrapolated), plasma clearance (Cl), terminal half-life (t½), terminal rate constant (kz), mean residence time (MRT) from time 0 to the last time point above the LLoQ, the MRT extrapolated to infinity (MRTINF), apparent volume of distribution at steady state (Vss), and apparent volume of distribution of the area during the elimination phase (Vz). The concentration at time 0 (C0) was calculated by log linear extrapolation using the first two time points after IV administration. The kz was determined using at least three time points. Pharmacokinetic data after oral administration included the AUC, AUCINF, MRT, MRTINF, t½, kz, plasma clearance per fraction of the dose absorbed (Cl/F), and apparent volume of distribution of the area during the elimination phase per fraction of the dose absorbed (Vz/F). The fraction of the dose absorbed (F) after oral amitriptyline was determined by dividing the oral AUCINF by the IV AUCINF. The maximum plasma concentration (CMAX) and time to maximum plasma concentration (tMAX) were determined directly from the data recorded after oral amitriptyline. The ratio of the nortriptyline AUCINF after oral amitriptyline to the nortriptyline AUCINF after IV amitriptyline (AUCINFOral/AUCINFIV) was determined. The accumulation ratio of amitriptyline after oral and IV dosing was estimated for 12 hour dosing intervals (s = 12 hours) with the following equation: 1 1
e
kzs The plasma concentration at 12 hours (C12) after oral administration was either determined directly from the data if greater than LLoQ or estimated from the last concentration greater than the LLoQ of the assay (Clast) at the time of Clast (Tlast) using the following equation: C12 ¼ ðClastÞ  e
kzð12 hours
TlastÞ Statistical testing of selected pharmacokinetic parameters was conducted in SigmaStat Version 12.0 (Systat Software, Inc., CA, USA) using a Wilcoxon signed-rank test (Powers 1990). A p-value of <0.05 was considered to indicate statistical significance.

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Amitriptyline pharmacokinetics in dogs C Norkus et al.

Results Orally administered amitriptyline was well tolerated by the dogs and no adverse effects were observed. Mild to moderate sedation, ataxia, transient tachycardia, polyphagia and vomiting were observed after IV administration for up to 3 hours. The mean oral and IV amitriptyline dose was 4.0 mg kg
1 administered as amitriptyline hydrochloride. The potency of the IV amitriptyline solution for injection was confirmed by LC/MS analysis (after storage at
70 °C) and was within acceptable variability of the assay (15% of the stated concentration; actual range: 87–97%). After IV amitriptyline administration, the C0 was 916.8 ng mL
1 with a terminal half-life of 10.92 hours and a mean residence time of 5.55 hours (Fig. 1 & Table 1). Plasma clearance was 16.9 mL minute
1 kg
1. The mean Vss and Vz were 9.01 and 16.77 L kg
1, respectively. The mean estimated accumulation ratio with a 12 hour dosing interval (AR12) was 1.96. The mean oral bioavailability of amitriptyline was 6% (range: 3–11%). The mean CMAX of amitriptyline was 27.4 ng mL
1 (range: 12.7–84.9 ng mL
1) and occurred at 1 hour. Orally administered amitriptyline was quickly eliminated with a 4.33 hour mean terminal half-life and 4.05 hour mean residence time (Fig. 1 & Table 2). The terminal halflives of amitriptyline did not differ statistically after IV and oral administration, respectively (p = 0.063). The mean plasma concentration at 12 hours (C12) was 4.6 ng mL
1. Only one animal had a C12 (9.3 ng mL
1) greater than the LLoQ of the assay

Figure 1 Plasma concentrations of amitriptyline after oral and intravenous (IV) administration of amitriptyline hydrochloride (mean dose: 4.0 mg kg
1) to healthy Greyhound dogs (n = 5). 4

(5 ng mL
1). The calculated C12 for the remaining animals ranged from 1.7 to 5.2 ng mL
1. The estimated accumulation ratio with a 12 hour dosing interval (AR12) was 1.28. The mean CMAX of nortriptyline following IV amitriptyline administration was 13.5 ng mL
1 (range: 11.6–15.7 ng mL
1) at 4.92 hours. The mean terminal half-life of nortriptyline following IV amitriptyline was 9.62 hours (Fig. 2 & Table 3). Following orally administered amitriptyline, the metabolite nortriptyline reached a mean CMAX of 14.4 ng mL
1 (range: 8.9–33.7 ng mL
1) at 2.04 hours (Fig. 3 & Table 4). Nortriptyline was eliminated with a 6.20 hour mean terminal half-life. The AUCINFOral to AUCINFIV ratio of nortriptyline was 61% (range: 38–88%). Discussion To the authors’ knowledge, this is the first study to report the pharmacokinetics of IV and oral amitriptyline and its metabolite nortriptyline in dogs. The non-compartmental pharmacokinetic analysis used in this study is a well-established pharmacokinetic modeling approach (Gabrielsson & Weiner 1997; Riviere 1999). Non-compartmental analysis is the preferred method for determining pharmacokinetic parameters including the AUC, TMAX, CMAX, volumes of distribution and clearance because it requires fewer assumptions than model-based approaches (Gillespie 1991; Gabrielsson & Weiner 2012). Additionally, non-compartmental methods have been preferred by the US Food and Drug Administration for the determination of the pivotal parameters of bioequivalence (AUC and CMAX) because these methods use raw data and do not require a specific model to fit the data, which may bias parameters based on the fit of regression curves (Anon 2002). As the primary purpose of the present study was to describe pharmacokinetic parameters including the AUC, TMAX and CMAX, volumes of distribution and clearance non-compartmental methodologies were used. The initial plasma samples were collected at 10 and 20 minutes after the IV injection. As the pharmacokinetic parameter C0 is determined by log linear regression of the first two time points, it is possible that C0 may have been underestimated. The time points for this study were chosen based on our ability to accurately and precisely collect samples after drug administration with the personnel and facilities available, but are a potential limitation for

© 2015 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia

Amitriptyline pharmacokinetics in dogs C Norkus et al. Table 1 Non-compartmental pharmacokinetic parameters of amitriptyline after intravenous administration of amitriptyline hydrochloride (mean dose: 4.0 mg kg
1) in five healthy Greyhound dogs

Parameter

Minimum

Median

Maximum

Geometric mean

Harmonic mean

AUC (hour 9 ng mL
1) AUCINF (hour 9 ng mL
1) AUCextrapolated (%) C0 (ng mL
1) Cl (mL minute
1 kg
1) T½ (hours) kz (1 hour
1) MRT (hours) MRTINF (hours) Vss (L kg
1) Vz (L kg
1) AR12 Dose (mg kg
1)

2580.7 2845.9 3.5 629.0 11.8 7.88 0.0386 5.09 6.69 6.74 11.46 1.53 3.9

3213.0 3350.6 5.3 860.6 17.5 10.66 0.0650 5.61 7.46 8.52 14.91 1.85 4.0

4510.6 5187.3 13.0 1657.8 20.3 17.98 0.0879 5.88 12.42 14.82 30.95 2.70 4.2

3218.7 3495.0 6.7 916.8 16.9 11.43 0.0606 5.55 8.86 9.01 16.77 1.96 4.0

3158.0 3422.1 5.8 868.1 16.6 10.92 0.0578 5.54 8.57 8.72 15.88 1.92 4.0

AUC, area under the curve from time 0 to the last time point above the analytical lower limit of quantification; AUCINF, AUC extrapolated to infinity; AUCextrapolated, % AUC extrapolated to infinity; C0, concentration extrapolated to time 0; Cl, plasma clearance; T½, terminal halflife; kz, terminal rate constant; MRT, mean residence time from time 0 to last time point above the LLoQ; MRTINF, MRT extrapolated to infinity; Vss, apparent volume of distribution at steady state; Vz, apparent volume of distribution of the area during the elimination phase; AR12, accumulation ratio with every 12 hour administration. Dose is expressed as amitriptyline hydrochloride.

Table 2 Non-compartmental pharmacokinetics of amitriptyline after oral administration of amitriptyline hydrochloride (mean dose: 4.0 mg kg
1) in five healthy Greyhound dogs

Parameter

Minimum

Median

Maximum

Geometric mean

Harmonic mean

AUC (hour 9 ng mL
1) AUCINF (hour 9 ng mL
1) AUCextrapolated (%) CMAX (ng mL
1) Cl/F (mL minute
1 kg
1) T½ (hours) λz (1 hour
1) MRT (hours) MRTINF (hours) TMAX (hours) Vz/F (L kg
1) F (%) AR12 C12 (ng mL
1) C12ss (ng mL
1) Dose (mg kg
1)

76.9 109.4 14.3 12.7 108.3 2.45 0.067 3.41 4.82 0.17 89.7 3 1.03 1.7 1.8 4.0

104.5 170.0 26.9 26.0 349.1 4.98 0.139 3.69 8.03 1.00 116.7 6 1.23 5.1 6.4 4.0

472.9 557.0 44.6 84.9 549.9 10.32 0.283 6.49 11.38 3.00 225.8 11 1.81 9.3 16.9 4.1

136.5 196.0 25.2 27.4 303.5 4.87 0.142 4.05 7.77 1.00 128.0 6 1.28 4.6 5.9 4.0

116.3 172.9 23.0 23.0 254.4 4.33 0.126 3.95 7.40 0.57 121.6 5 1.26 3.9 4.5 4.0

AUC, area under the curve from time 0 to the last time point above the analytical lower limit of quantification; AUCINF, AUC extrapolated to infinity; AUCextrapolated, % AUC extrapolated to infinity; CMAX, maximum plasma concentration; Cl/F, clearance per fraction absorbed; T½, terminal half-life; kz, terminal rate constant; MRT, mean residence time from time 0 to last time point above the LLoQ; MRTINF, MRT extrapolated to infinity; TMAX, time of CMAX; Vz/F, Vz per fraction absorbed; F, fraction of the dose absorbed; AR12, accumulation ratio with every 12 hour administration; C12, concentration at 12 hours after administration; C12ss, concentration at 12 hours at steady state with every 12 hour administration. Dose is expressed as amitriptyline hydrochloride.

the calculation of C0. C0 might be underestimated if plasma concentrations declined rapidly in the first 10 minutes.

The calculated terminal half-life was numerically smaller after oral administration than after IV administration, but did not differ significantly

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Amitriptyline pharmacokinetics in dogs C Norkus et al.

Figure 2 Plasma concentrations of nortriptyline after intravenous administration of amitriptyline hydrochloride (mean dose: 4.0 mg kg
1) in healthy Greyhound dogs (n = 5).

(p = 0.063). As the difference in the terminal halflives was not statistically significant, the numerical differences may reflect simple random variability. It is possible that the concentrations after oral administration were too low to allow the detection of a deep compartment elimination, which may have occurred at very low concentrations below the analytical sensitivity of the assay (5 ng mL
1). If this is the case, then the AUC for oral administration may be underestimated, leading to an underestimation of oral bioavailability. It was hypothesized that even if a deep compartment elimination were to occur after oral administration and were to be missed in the study, it would have insignificant and irrelevant effects on the

calculated oral bioavailability. The impact of a deep compartment elimination phase that may have occurred below the LLoQ was assessed using data simulations. As a worst case scenario, the IV terminal rate constant representing a supposed deep compartment elimination was modeled as a deep compartment terminal rate constant after oral administration and hence the extrapolated portion of the oral AUC was extrapolated using the IV kz, from the Clast after oral administration. These simulations represent a worst case scenario for a data bias such that the deep compartment elimination started immediately after the last plasma concentrations above the LLoQ of the assay. The resultant simulated data showed a slight change in the calculated F (mean 8%; range: 4–12%) incorporating a supposed deep compartment elimination, which did not differ significantly (p = 0.063) from the actual calculated F (mean 6%; range: 3–11%). Therefore, even if a deep compartment elimination occurs after oral administration and was not detected in this study, simulations suggest it would contribute minimally to overall oral bioavailability, would not significantly affect oral bioavailability, and would be clinically irrelevant. Similarly, the effects of a deep compartment elimination on potential accumulation after multiple oral doses were assessed. It was hypothesized that a supposed deep compartment elimination occurring at such low concentrations would be insignificant and clinically irrelevant to potential drug steadystate minimum plasma concentrations (C12ss) with a 12 hour dosing interval. The predicted C12 at

Table 3 Non-compartmental pharmacokinetic parameters of nortriptyline after intravenous administration of amitriptyline hydrochloride (mean dose: 4.0 mg kg
1) in five healthy Greyhound dogs

Parameter

Minimum

Median

Maximum

Geometric mean

Harmonic mean

AUC (hour 9 ng mL
1) AUCINF (hour 9 ng mL
1) AUCextrapolated (%) CMAX (ng mL
1) T½ (hours) λz (1 hour
1) MRT (hours) MRTINF (hours) TMAX (hours)

105.0 192.8 23 11.6 6.51 0.053 5.57 10.97 3.00

125.1 250.2 46 13.2 10.38 0.067 5.93 16.46 6.00

266.6 347.8 58 15.7 13.07 0.107 9.75 20.49 6.00

143.1 253.4 40 13.5 9.90 0.070 6.42 15.63 4.82

136.8 247.5 38 13.4 9.62 0.068 6.30 15.30 4.62

AUC, area under the curve from time 0 to the last time point above the analytical lower limit of quantification; AUCINF, AUC extrapolated to infinity; AUCextrapolated, % AUC extrapolated to infinity; CMAX, maximum plasma concentration; T½, terminal half-life; kz, terminal rate constant; MRT, mean residence time from time 0 to last time point above the LLoQ; MRTINF, MRT extrapolated to infinity; TMAX, time of CMAX.

6

© 2015 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia

Amitriptyline pharmacokinetics in dogs C Norkus et al.

Figure 3 Plasma concentrations of nortriptyline after oral amitriptyline hydrochloride (mean dose: 4.0 mg kg
1) in healthy Greyhound dogs (n = 5).

steady state for oral dosing using the IV AR12, assuming a deep compartment elimination, would give a mean value of 13 ng mL
1 (range: 5.8– 21.5 ng mL
1), whereas the predicted C12 based on the actual data in the study gave a mean value of 5.9 ng mL
1 (range: 1.8–16.9 ng mL
1), which is not statistically different (p = 0.063). The small magnitude at such low concentrations is clinically irrelevant. However, multiple-dose studies achieving steady-state plasma concentrations should be performed to confirm the simulations. The lack of potential differences in both accumulation and oral bioavailability with a theoretical deep compartment elimination can be attributed to the fact that elimination occurs at therapeutically

irrelevant concentrations after oral administration (Toutain & Bousquet-M
elou 2004). The lack of relevance derives from the fact that the vast majority of drug is eliminated prior to the theoretical deep compartment elimination phase, which thus has minimal, insignificant and clinically irrelevant effects on predicted oral bioavailability or drug accumulation with simulated multiple doses. However, studies administering multiple doses of amitriptyline are indicated to confirm these speculations. Previous work in dogs suggested similar peak plasma concentrations of amitriptyline occurred in dogs compared with humans on a mg kg
1 basis (Kukes et al. 2009). Canine subjects in the study by Kukes et al. (2009) achieved substantially higher plasma concentrations of amitriptyline (ranging from 126.1  8.6 to 149.7  17.0 ng mL
1) following a 3.57 mg kg
1 orally administered dose of amitriptyline than were measured in this study (mean CMAX: 27.4 ng mL
1) using a similar dose. Several possible explanations for this variation exist. First, Kukes et al. (2009) used mixed-breed dogs, whereas this study utilized Greyhound dogs. Greyhound dogs have previously been shown to have unique pharmacokinetic profiles for thiobarbiturates and propofol, and demonstrate slower clearance than do other canine breeds; however, to date no significant pharmacokinetic differences with other drugs have been documented (Sams et al. 1985; Robinson et al. 1986; Zoran et al. 1993; KuKanich & Nauss 2012). If a pattern of decreased clearance similar to that of thiobarbiturates and propofol were

Table 4 Non-compartmental pharmacokinetic parameters of nortriptyline after oral administration of amitriptyline hydrochloride (mean dose: 4.0 mg kg
1) in five healthy Greyhound dogs

Parameter

Minimum

Median

Maximum

Geometric mean

Harmonic mean

AUC (hour 9 ng mL
1) AUCINF (hour 9 ng mL
1) AUCextrapolated (%) CMAX (ng mL
1) T½ (hours) kz (1 hour
1) MRT (hours) MRTINF (hours) TMAX (hours) AUCINFOral/AUCINFIV (%)

59.9 114.8 23 8.9 5.48 0.099 3.87 8.44 1.00 38

65.8 125.8 47 11.7 6.41 0.108 4.08 10.22 3.00 65

234.2 306.0 51 33.7 7.03 0.126 5.38 11.30 4.00 88

92.0 153.9 37 14.4 6.23 0.111 4.40 9.87 2.05 61

81.9 145.6 35 13.1 6.20 0.111 4.37 9.81 1.71 58

AUC, area under the curve from time 0 to the last time point above the analytical lower limit of quantification; AUCINF, AUC extrapolated to infinity; AUCextrapolated, % AUC extrapolated to infinity; CMAX, maximum plasma concentration; T½, terminal half-life; kz, terminal rate constant; MRT, mean residence time from time 0 to last time point above the LLoQ; MRTINF, MRT extrapolated to infinity; TMAX, time of CMAX; AUCINFOral/AUCINFIV, ratio of the nortriptyline AUCINF after oral amitriptyline to the nortriptyline AUCINF after IV amitriptyline.

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Amitriptyline pharmacokinetics in dogs C Norkus et al.

to occur with amitriptyline, then Greyhounds would be expected to show much higher oral bioavailability than other dog breeds as a result of a lower first pass metabolism effect. It is possible that there are unrecognized breed-specific differences in the metabolism of this drug within the canine species. A lower amount of drug absorbed or higher first pass metabolism may have occurred in the Greyhounds. Unfortunately, there are no reports of IV amitriptyline pharmacokinetics in other dog breeds available for comparison. Further studies incorporating multiple dog breeds or a population pharmacokinetic study may be able to differentiate potential breedspecific differences in the pharmacokinetics of amitriptyline in dogs, if they exist. Another consideration is that amitriptyline was administered to non-fasted subjects in this study to mimic clinical conditions that would maximize client compliance. Kukes et al. (2009) administered amitriptyline to fasted subjects with a ‘small amount of curd cheese’. A significant drug–food interaction could be present in dogs and thus the absorption of the drug in the present study may have been limited. To date, no food effect on drug absorption has been identified with amitriptyline in humans and therefore this phenomenon would be specific to the canine species. Kukes et al. (2009) used different tablet formulations of amitriptyline produced outside the USA, whereas this study used an FDA-approved product. It is possible that these different formulations are not bioequivalent in dogs and result in different levels of oral bioavailability. In order for a drug to be absorbed from a solid dosage form, it must first disintegrate and then dissolve within the gastrointestinal tract. For example, a study assessing the effects of oral sucralfate in a capsule formulation in Beagles showed the effective release of sucralfate (Steiner et al. 1982), but a study of sucralfate in a tablet formulation in Greyhounds showed that large fragments of tablet were passed intact in the feces, indicating poor disintegration (KuKanich & KuKanich 2014). Amitriptyline tablet fragments were not grossly observed in the feces of the dogs in this study. Further studies comparing the administration of amitriptyline as a solution or suspension for oral administration with the administration of the tablet formulation are indicated to assess the potential effect of tablet disintegration or dissolution on the oral bioavailability of the drug. Analytical methodology may bias results. This study used LC/MS as the method of analysis, 8

whereas Kukes et al. (2009) used high-performance liquid chromatography (HPLC) without determining nortriptyline or other amitriptyline metabolite plasma concentrations. Hucker et al. (1977) evaluated urinary metabolites of amitriptyline in the dog and identified at least six metabolites, including two dihydrodiols of amitriptyline, amitriptyline N-oxide, 10-hydroxyamitriptyline, 10-hydroxynortriptyline and nortriptyline. A limitation of any HPLC analysis is that co-elution of amitriptyline and amitriptyline metabolites may occur and may be incorrectly identified as higher amitriptyline plasma concentrations if amitriptyline metabolites co-elute with amitriptyline. Kukes et al. (2009) did not report any assessment for interferences from amitriptyline metabolites and therefore it is unclear if their HPLC assay accurately quantified amitriptyline plasma concentrations. The low plasma concentrations measured after the 4 mg kg
1 targeted dose of oral amitriptyline in Greyhounds may be attributable to a high first pass metabolism or poor gastrointestinal absorption. It is unclear if first pass metabolism, poor absorption or a combination thereof contributed to the low oral bioavailability (6%). The mean nortriptyline AUCINF Oral/AUCINFIV was 61%, despite administration of the same dose orally and IV, which suggests that either a smaller portion of the amitriptyline was available for metabolism to nortriptyline after oral administration (lower absorption) or the metabolism pathways for the oral and IV routes of administration differ. However, the AUCINFOral/AUCINFIV of nortriptyline after amitriptyline was much higher than the amitriptyline oral bioavailability (6%), which suggests that first pass metabolism to other metabolites that were not quantified may have occurred. According to Hucker et al. (1977), the major metabolite of amitriptyline in the urine of two Beagle dogs was 10-hydroxyamitriptyline. No reference standard for 10-hydroxyamitriptyline was available at the time of analysis and assessment of the concentrations or ratio of this metabolite after oral and IV administration of amitriptyline was not feasible. Therefore, the low oral bioavailability of amitriptyline in dogs may reflect both incomplete absorption and first pass metabolism to inactive metabolites and hence additional investigation to identify the relative contributions to the low bioavailability is required. Further research is also indicated to evaluate whether the lower than expected plasma levels are a result of breed variation, drug–food effect, inherently low oral bioavailability

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Amitriptyline pharmacokinetics in dogs C Norkus et al. arising from poor absorption or first pass metabolism, poor disintegration or dissolution of the tablet, and whether a proportional increase in plasma concentration occurs with a higher oral dose. In conclusion, amitriptyline and nortriptyline plasma concentrations were lower than expected after oral administration to Greyhound dogs. These results suggest further studies to assess the efficacy of the currently recommended doses of oral amitriptyline of 1–4 mg kg
1 in dogs (Cashmore et al. 2009; Plumb 2011) are required. Studies assessing possible causes of low plasma concentrations after oral administration should be conducted, with the eventual goal of testing the antinociceptive effects of orally administered amitriptyline in dogs to determine if it is effective. Acknowledgements The authors declare no conflict of interest. References Anon (2002) Guidance for Industry: Bioequivalence Guidance. US Department of Health and Human Services, Food and Drug Administration, Center for Veterinary Medicine, USA. Antkiewicz-Michaluk L, Romanska I, Michaluk J et al. (1991) Role of calcium channels in effects of antidepressant drugs on responsiveness to pain. Psychopharmacology 105, 269–274. Cai Z, McCaslin PP (1992) Amitriptyline, desipramine, cyproheptadine and carbamazepine, in concentrations used therapeutically, reduce kainate and N-methyl-Daspartate-induced intracellular Ca2+ levels in neuronal culture. Eur J Pharmacol 219, 53–57. Cashmore RG, Harcourt-Brown TR, Freeman PM et al. (2009) Clinical diagnosis and treatment of suspected neuropathic pain in three dogs. Aust Vet J 87, 45–50. Dharmshaktu P, Tayal V, Kalra BS (2012) Efficacy of antidepressants as analgesics: a review. J Clin Pharmacol 52, 6–17. Eschalier A, Montastruc JL, Devoize JL et al. (1981) Influence of naloxone and methysergide on the analgesic effect of clomipramine in rats. Eur J Pharmacol 74, 1–7. Gabrielsson J, Weiner D (1997) Non-compartmental analysis. In: Pharmacokinetic and Pharmacodynamic Data Analysis: Concepts and Applications (2nd edn). Gabrielsson J, Weiner D (eds). Swedish Pharmaceutical Press, Sweden. pp. 139–153. Gabrielsson J, Weiner D (2012) Non-compartmental analysis. Methods Mol Biol 929, 377–389. Galeotti N, Ghelardini C, Bartolini A (2001) Involvement of potassium channels in amitriptyline and clomipramine analgesia. Neuropharmacology 40, 75–84.

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