Pharmacokinetics of a high-concentration formulation of buprenorphine (Simbadol) in

male dogs

Jeremy Dustin Hansford

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in

partial fulfillment of the requirements for the degree of

Master of Science

In

Biomedical and Veterinary Sciences

Committee Chair: Natalia Henao-Guerrero

Committee Member: Jennifer L. Davis

Committee Member: Vaidehi V. Paranjape

April 19, 2021

Blacksburg, Virginia

Keywords: biological availability, buprenorphine, dogs, nonlinear mixed-effects modeling,

pharmacokinetics

Pharmacokinetics of a high-concentration formulation of buprenorphine (Simbadol) in male dogs

Jeremy Dustin Hansford

ABSTRACT (ACADEMIC)

Objective To describe the pharmacokinetics of buprenorphine in dogs following administration

of a high-concentration formulation of buprenorphine.

Study design Prospective, randomized, crossover study.

Animals A total of six healthy male intact Beagle dogs, 9–13 months of age and weighing 10.3 ±

1.4 kg (mean ± standard deviation).

Methods Dogs were randomized to be administered buprenorphine (0.12 mg kg−1; Simbadol, 1.8

mg mL−1) via the intravenous (lateral saphenous) or subcutaneous (dorsal interscapular) route

followed by the alternative route of administration after a 14 day interval. Blood was sampled

before administration and at set times up to 72 hours after injection. Plasma buprenorphine

concentration was measured using liquid chromatography–tandem mass spectrometry.

Results A 3-compartment model with zero or biphasic rapid and slow first order input in

(intravenous or subcutaneous data, respectively) and first-order elimination from the central

compartment best fitted the data. The rapid first order input accounted for 63% of the dosage

absorption. Typical values (% interindividual variability) for the three compartment volumes

were 900 (33), 2425 (not estimated) and 6360 (28) mL kg−1. The metabolic and two distribution

clearances were 25.7 (21), 107.5 (74) and 5.7 (61) mL minute−1 kg−1. The absorption half-life for

the fast absorption phase was 8.9 minutes with a 0.7 (103) minute delay. The absorption half-life

for the slow absorption phase was 347 minutes with a 226 (42) minutes delay. Median (range)

bioavailability calculated from noncompartmental analysis was 143 (80–239) %. Calculated

terminal half-life was 963 minutes.

Conclusions and clinical relevance The high-concentration formulation of buprenorphine

administered subcutaneously had a large volume of distribution and a rapid absorption phase

followed by slower, delayed absorption. The high estimate of bioavailability should be

interpreted with caution as values above 100% are most commonly related to experimental

issues.

Pharmacokinetics of a high-concentration formulation of buprenorphine (Simbadol) in male dogs

Jeremy Dustin Hansford

ABSTRACT (GENERAL AUDIENCE)

Veterinary pain management is a growing industry with increased knowledge on the

degree of pain animals experience. Buprenorphine is a partial μ-agonist opioid and Schedule III

controlled substance in the United States, indicating it is less habit-forming than Schedule II

opioids, such as morphine. There is an FDA-approved long-acting formulation of buprenorphine

available for cats (Simbadol®), and it is produced specifically for veterinary use. The

combination of Schedule III plus a veterinary-specific formulation allows easier and more

reliable access to veterinarians; thus, it has been used clinically off-label in dogs despite the lack

of empirical information.

The present study is descriptive in nature. Six healthy male Beagle dogs were utilized in a

prospective, randomized, crossover study. Each was anesthetized and a central venous catheter

placed subcutaneously in the right external jugular vein. Once recovered, 0.12mg kg-1

Simbadol® buprenorphine was given subcutaneously or intravenously and blood samples

collected at multiple time points from 1 minute to 72 hours for determination of plasma

concentration. Following completion of blood collection, the catheters were removed and the

dogs allowed a 14-day washout interval. The process was then repeated with the left external

jugular vein utilized for catheter placement and each dog receiving the opposite treatment route

of administration from the first period.

Drug absorption following subcutaneous administration was determined. There was a

rapid initial absorption phase accounting for 63% of total drug absorbed, following by a slower

delayed absorption phase. Overall absorption was 131%, potentially suggesting differences in

drug metabolism among the different routes.

Side effects varied among dogs, with undetectable to marked sedation occurring in both

treatment groups. Hypersalivation and whining were fairly common, tending to occur in the

same dogs in both treatment phases. All dogs had a reduced appetite for the first 24 hours in the

first phase of the study but not the second phase.

The high-concentration formulation of buprenorphine administered subcutaneously in

dogs was characterized by a biphasic absorption with high bioavailability. Side effects were

noted in some dogs, regardless of route. This study provides preliminary data on the disposition

of Simbadol® in dogs. Data is still needed to determine effective therapeutic concentrations so

appropriate doses can be determined.

vi

ACKNOWLEDGEMENTS

The authors thank Dr. Joao HN Soares for his contribution to early study planning. This study

was funded by a grant from the Center for Companion Animal Health, School of Veterinary

Medicine, University of California (no. 2018-36-F).

vii

ATTRIBUTION

Three co-authors assisted in collection of samples, analysis of data, writing and revision of the

manuscript. A brief description of each person’s contribution is included here:

Jeremy Hansforda: execution of the study, data acquisition, manuscript writing and revision

Natalia Henao-Guerreroa: conception, design and execution of the study, data acquisition,

manuscript writing and revision

Marcela L Machadob: execution of the study, data acquisition, manuscript revision

Bruno Pypendopc: conception, design, data management, statistical analysis, manuscript writing

and revision

aDepartment of Small Animal Clinical Sciences, Virginia Maryland College of Veterinary

Medicine, Blacksburg, VA, USA

bWilliam R Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine,

University of California–Davis, Davis, CA, USA

cDepartment of Surgical and Radiological Sciences, School of Veterinary Medicine, University

of California–Davis, Davis, CA, USA

This article was published in Veterinary Anaesthesia and Analgesia, 48, Hansford J, Henao-

Guerrero N, Machado ML et al., Pharmacokinetics of a high-concentration formulation of

buprenorphine (Simbadol) in male dogs, 509-516, Copyright Elsevier (2021).

DOI: https://doi.org/10.1016/j.vaa.2021.04.003

viii

TABLE OF CONTENTS

Abstract (Academic) – ii

Abstract (General Audience) – iv

Acknowledgements – vi

Attribution – vii

I. Introduction – 1

II. Materials and Methods – 2

III. Results – 8

IV. Discussion – 9

V. Conclusion – 15

VI. Figures 1 and 2 – 16

VII. Table 1 – 18

VIII. References – 20

IX. Appendix SA – 24

1

I. INTRODUCTION

Opioids are the hallmark of veterinary pain control, yet there is an increasing shortage of

opioid availability, partly from manufacturing complications and from human diversion.

Buprenorphine, a partial µ-opioid receptor agonist, has been used in dogs for analgesia both pre-

and postoperatively. It is a Schedule III narcotic substance within the United States, with less

potential for abuse than Schedule I and II narcotics. Although no buprenorphine formulation is

approved by the US Food and Drug Administration (FDA) for usage in dogs, buprenorphine is

administered intravenously (IV), intramuscularly (IM) and subcutaneously (SC) at 6–8 hour

intervals for analgesia and sedation.

The pharmacokinetics of buprenorphine in cats has been described following both IV

(Steagall et al. 2013; Doodnaught et al. 2017) and SC (Steagall et al. 2013; Taylor et al. 2016;

Doodnaught et al. 2017) routes, but there is a comparative paucity of information in dogs.

Pharmacokinetics of IV buprenorphine, both at high (Garrett & Chandran 1990) and common

dose rates administered clinically (Krotscheck et al. 2008; Barbarossa et al. 2017) have been

reported. Studies of the analgesic effect of buprenorphine administered SC have been published

(Moll et al. 2011; Nunamaker et al. 2014). However, at the time of writing, there is only one

report of SC pharmacokinetics of a high-dose extended-release buprenorphine in dogs (Barletta

et al. 2018). This formulation is approved for mice and rats, requiring separate ordering and off-

label usage in dogs. A veterinary-specific, FDA-approved formulation for cats is also available

(Simbadol; Zoetis US, NJ, USA) with one published report of the pharmacokinetics in dogs

(Steagall et al. 2020).

The objective of the present study was to determine the pharmacokinetics of a high-

concentration formulation of buprenorphine in dogs, administered IV and SC.

2

II. MATERIALS AND METHODS

Animals:

The present study was approved by the Virginia Tech Institutional Animal Care and Use

Committee (no. 18-178). A total of six healthy intact male Beagle dogs (aged 9–13 months,

weighing 10.3 ± 1.4 kg) were utilized in a randomized crossover design study. All were deemed

healthy based on physical examination, complete blood count and serum chemistries. Dogs were

housed with toys available within the university animal research facility and allowed to

acclimate to the laboratory environment for 3 days before starting the study. When possible, dogs

were housed in groups. Food was withheld for 12 hours prior to anesthesia; water was offered ad

libitum. Each dog completed both treatments, with 14 days between treatments. Dogs were

observed for sedation and adverse effects during the sampling periods.

Instrumentation:

Isoflurane 5% (Fluriso; VetOne, ID, USA) in oxygen was administered by facemask until

a plane of anesthesia sufficient for tracheal intubation was reached. After orotracheal intubation,

each dog was positioned in left lateral recumbency and connected to a circle system delivering

isoflurane in oxygen (BleaseFocus Anesthesia Machine; Spacelabs Healthcare, WA, USA). Eyes

were lubricated (Puralube Vet Ointment; Dechra Veterinary Products LLC, KS, USA) and dogs

were instrumented with monitoring equipment. Pulse rate (PR) from pulse oximeter, respiratory

rate (fR) from capnometer, oscillometric blood pressure, esophageal temperature, pulse oximetry

(% hemoglobin oxygen saturation), continuous electrocardiogram (ECG), partial pressure of end-

tidal carbon dioxide (PECO2), end-tidal isoflurane concentration (FEIso) and inspired oxygen

concentration were monitored throughout each anesthetic episode (Datex-Ohmeda S/5

3

Anesthesia Monitor; GE Healthcare, CT, USA). PR, fR and blood pressure were recorded on a

paper anesthetic log every 5 minutes, and the remaining monitoring variables were recorded

every 15 minutes. All dogs breathed spontaneously, the FEIso was adjusted as needed to

maintain an appropriate depth of anesthesia, and lactated Ringer’s solution (LRS; Baxter

Healthcare Corp., IL, USA) was administered at 3 mL kg−1 hour−1 throughout anesthesia. If

mean arterial pressure (MAP) decreased to < 60 mmHg, LRS (10 mL kg−1) was infused IV over

10 minutes and FEIso was reduced if possible. If MAP remained < 60 mmHg following these

adjustments, a continuous rate IV infusion of dopamine (5–15 μg kg−1 minute−1; Hospira Inc., IL,

USA) was administered. An underbody warm water blanket (T/Pump Classic; Gaymar Industries

Inc., NY, USA) was provided to maintain an esophageal temperature 36.7–38.3 °C during the

procedure.

The hair over the right lateral saphenous vein was clipped and the skin aseptically

prepared with ethyl alcohol 70% and povidone iodine 7.5% scrub (Pivodine Scrub; VetOne). A

20 gauge, 3.2 cm peripheral IV catheter (Jelco; Smiths Medical International Ltd, UK) was

inserted in the vein and secured with porous medical adhesive tape (Johnson & Johnson, NJ,

USA) circumferentially around the limb. The hair over the right external jugular vein was

clipped from the thoracic inlet to the caudal aspect of the ramus of the mandible, extending

ventrally and dorsally to near respective midlines. The skin was aseptically prepared in the same

manner as for the lateral saphenous vein. A 3–4 cm incision was made through the skin over the

jugular vein and subcutaneous tissue separated allowing direct view of the external jugular vein.

A 5 Fr, 13 cm double-lumen central venous catheter (MILA International Inc., KY, USA) was

inserted into the right external jugular vein and the wing sutured to the dermis, effectively

burying the catheter. An injection cap (PRN cap; B. Braun Medical Inc., PA, USA) was attached

4

to each integrated extension set of the catheter. A 2 cm incision was made through the skin along

dorsal midline of the cranial cervical region, and Kelly hemostatic forceps were used to disrupt

the SC tissue from dorsal midline down to the catheter, creating a tunnel. The integrated

extension sets of the catheter were then grasped and retracted through this tunnel, effectively

burying the entire catheter SC in the right lateral neck region. The dorsal midline and the initial

skin incision over the jugular vein were then closed using an intradermal pattern, avoiding

external sutures. Upon conclusion of central venous catheter placement, the lateral saphenous

catheter was removed and the dog recovered from anesthesia. No bandages or E-collars were

required given the SC placement and lack of external sutures. For the second treatment,

catheterization technique was identical, except that a 4 Fr, 15 cm single-lumen central venous

catheter (MILA International Inc.) was inserted into the left external jugular vein.

Study protocol:

Dogs were randomly assigned via random-number generator (Microsoft Excel Version

16; Microsoft, WA, USA) to be administered buprenorphine (0.12 mg kg−1; Simbadol, 1.8 mg

mL−1; Zoetis US) either SC (dorsal interscapular space) or IV the day after central venous

catheter placement for the first treatment. Thus, three treatments for each route were

administered, and 14 days later the alternate treatment was administered.

On the morning of the IV buprenorphine treatment, a lateral saphenous catheter was

inserted aseptically for drug administration. Buprenorphine was administered as a bolus over 2

seconds or less, followed by flushing with 2 mL sterile saline. The catheter was then removed.

Dogs assigned to the SC treatment were injected in the dorsal midline interscapular space with a

22 gauge, 2.5 cm needle (Monoject; Covidien LLC, MA, USA. Central venous blood (2 mL) was

5

collected before administration of buprenorphine and at 1, 2, 4, 8, 15, 30, 60, 120, 240, 480, 720,

1440, 2160, 2880, 3600 and 4320 minutes after administration. For each sample collection, the

skin over the injection port of the central venous catheter extension was cleaned with ethyl

alcohol 70%. Next, a 3 mL syringe containing 1 mL 0.9% saline attached to a 22 gauge needle

was used to broach the injection port through the overlying skin. Following puncture of the

injection cap, a 1 mL waste sample was removed, then the syringe was detached and a separate

empty 3 mL syringe was attached to withdraw 2 mL of blood. The original waste sample was

returned to each dog and the catheter was flushed with 2 mL 0.9% saline. The blood sample was

transferred to a tube containing ethylenediaminetetraacetic acid (K2 EDTA BD Vacutainer; BD

Medical, NJ, USA). After the longer-interval time points (480, 720, 1440, 2160, 2880 and 3600

minutes), the catheter was flushed with 0.4 mL heparinized saline (5 IU mL−1) to reduce clotting.

Blood samples were centrifuged at 4 °C for 10 minutes at 1230 g (Centra CL3-R; Thermo Fisher

Scientific, MA, USA) within 15 minutes of sampling. Plasma was then transferred to cryotubes

(Cryogenic vials; Corning Incorporated, NY, USA) using plastic sampling pipettes (Fisherbrand

transfer pipets; Fisher Scientific Company LLC, MA, USA), and frozen at –80 °C until analysis

for buprenorphine concentration. After the final sample was collected, each dog was

reanesthetized with isoflurane in oxygen as described previously. The central venous catheter

was removed by reopening the original lateral neck incision through which the catheter had been

placed. The skin incision was closed with absorbable suture material in an intradermal pattern.

Each dog was administered carprofen (4.4 mg kg−1; Ostifen; VetOne, Northern Ireland, UK) SC

over the dorsal lumbar spine and allowed to recover from anesthesia.

Buprenorphine assay:

6

Buprenorphine was quantified in protein-precipitated plasma samples using liquid

chromatography–tandem mass spectrometry according to a modification of a previously

published method (Yue et al. 2005). The assay was partially validated for dog plasma according

to the criteria set forth by the FDA Guidance to Industry on Bioanalytical Assay Validation. D-4

buprenorphine was used as the internal standard. The lower limit of quantification was 0.05 ng

mL−1. Accuracy (% nominal concentration) and imprecision (coefficient of variation) were

verified at 0.15, 5 and 40 ng mL−1 and ranged from 89 to 108% and from 1 to 9%, respectively.

Pharmacokinetic analysis:

Noncompartmental analysis was initially used to explore the data, obtain initial estimates

for the compartment models and estimate bioavailability following SC administration. One- (SC

data only), two- and three-compartment models with first-order elimination from the central

compartment were then fitted to the time-plasma buprenorphine concentration-time data in each

dog. Bolus input within the central compartment was specified for the IV models, and different

absorption models (first order with and without lag time, two first order absorption phases,

inverse gaussian and inverse gaussian with time-delay) within the central compartment were

tested for the data following SC administration. The best fitting individual models (based on

observation of the residual plots, -2log likelihood and Akaike’s information criterion) were used

to select the initial structure of, and obtain the initial parameter estimates for the final model. A

population model fitting the IV and SC data simultaneously using nonlinear mixed effect

modeling was developed, with the assumption that the volumes and clearances were identical for

IV and SC administration (Appendix SA). The best fitting model was selected based on

observation of the predicted versus observed data plots, the residual plots, the precision of the

7

parameters, the -2 log likelihood and Akaike’s information criterion. Additional parameters were

calculated from the parameters estimated by the model using standard pharmacokinetic equations

(Gabrielsson & Weiner 2016). All analyses were conducted using Phoenix WinNonlin and

Phoenix NLME Version 8.2 (Certara Inc., NJ, USA). Pharmacokinetic parameters are presented

as typical (population) value, precision (coefficient of variation) and % interindividual

variability.

8

III. RESULTS

All dogs completed both treatments. The central venous catheters remained patent and

accessible at all sample time points in all dogs in both treatments. Plasma buprenorphine

concentrations following IV and SC administration are presented in Figure 1. A three-

compartment model with bolus input (IV data) and a rapid first order absorption phase with a

short delay followed by a slow first order absorption phase with a longer delay (lag time; SC

data) into the central compartment best fitted the plasma buprenorphine concentration-time data

(Fig. 2), and typical values for its pharmacokinetic parameters are listed in Table 1. The model

code and goodness-of-fit plots are available in Appendix SA. Median (range) bioavailability

calculated by noncompartmental analysis was 143 (80–239) %.

Varying degrees of sedation, from none to marked, were observed lasting 0.5–8 hours.

Panting, whining and hyperptyalism were noted. No emesis was observed after buprenorphine

administration in any dog, although 4/6 dogs had reduced appetites the night following the SC

treatment and 2/6 following the IV treatment. No other adverse events were noted related to

administration of buprenorphine. Following completion of the study and removal of central

venous catheters, all dogs were castrated and adopted.

9

IV. DISCUSSION

The present report describes the pharmacokinetic profile of a high-concentration

formulation of buprenorphine administered IV or SC in six male Beagle dogs. A three-

compartment model described the data adequately. The disposition of buprenorphine was

characterized by a large volume of distribution and a moderate clearance, resulting in a terminal

half-life of 16 hours.

Bioavailability of buprenorphine following SC administration has been reported as dose-

dependent. At a dosage of 0.24 mg kg−1 SC in cats, the bioavailability of buprenorphine was 94%

with a biphasic absorption in the central compartment (Doodnaught et al. 2017). The initial

concentration peak occurred at 5 minutes followed by a slow continued uptake until 12 hours,

after which the plasma concentration began to steadily decline. In a previous study, at a lower

dosage of 0.02 mg kg−1, absorption was reported to be erratic, preventing the assessment of

bioavailability (Steagall et al. 2013). However, the time-mean plasma buprenorphine

concentration profile presented suggests that two absorption phases were present. In dogs, a low

dosage of 0.02 mg kg−1 achieved 40% bioavailability with a low peak plasma concentration and

erratic uptake (Steagall et al. 2020). The current study of a high dosage of buprenorphine (0.12

mg kg−1) showed a biphasic absorption as seen in cats, although the second absorption phase was

not obvious in some dogs based on observation of the time-concentration curves. The

bioavailability after SC administration in the present study largely exceeded 100%, both when

calculated by the ratios of the areas under the time-concentration curves obtained by

noncompartment analysis and estimated by the compartment model. The estimates of

bioavailability were much higher than the value (94%) found in cats for a high dosage of a high-

concentration formulation of buprenorphine (Doodnaught et al. 2017). A separate investigation

10

into a liposomal-encapsulated formulation of buprenorphine (0.2 mg kg−1) administered SC to

cats found absolute bioavailabilities that ranged from 63 to 258% (Johnson et al. 2017).

Similarly, a study in humans found that the SC administration of a depot buprenorphine

formulation resulted in absolute bioavailability ranging 129–172% depending upon the dosage

(Albayaty et al. 2017). The reason for bioavailability to be > 100% is not entirely clear, and this

estimate should be interpreted with caution. Values > 100% are most commonly the result of

experimental errors. In the present study, bioavailability exceeded 100% in three dogs but was <

100% in the other three dogs. The estimation of bioavailability assumes that there is no

difference in clearance between the two routes of administration. The same dogs were studied for

IV and SC administration in the present study, and the washout interval (14 days) was relatively

short, reducing the likelihood for large violations of the assumption of equal clearance. The

randomization was balanced (i.e., three dogs were administered either the IV or SC treatment

first), which should minimize a possible effect of the order of treatment (e.g., enzyme induction

or inhibition). Nevertheless, a difference in clearance contributing to the high bioavailability

cannot be completely ruled out.

Another possible explanation for a bioavailability greater than 100% is contamination of

the samples with blood containing a high concentration of buprenorphine following

subcutaneous administration. It has been shown that sampling blood in the vein draining the

extravascular administration site results in erroneously high values of bioavailability because the

drug concentration in the sample is higher than in a well-mixed sample obtained elsewhere

(Hedges et al. 2014). In the present study, the drug was administered subcutaneously in the

interscapular space. Venous drainage of this site is probably through the thoracodorsal vein,

connecting sequentially to the axillary vein, the subclavian vein, the brachiocephalic vein and the

11

external jugular (Pavletic 1980). However, drug concentrations in the samples higher than in a

well-mixed sample accounting for the high bioavailability is considered unlikely for the

following reasons: 1) the tip of the catheter may have been in the cranial vena cava rather than

the jugular vein in a majority of dogs; 2) the samples would be diluted with blood draining areas

other than the administration site; and 3) because the administration site was on or close to

midline the drug would have drained to both sides of the body. Although the catheters were

changed from a double to a single lumen for the second treatment as a refinement and to reduce

SC tissue disruption needed for placement, the lengths of 13 and 15 cm, respectively, should

have resulted in similar locations of the distal tip. Nonetheless, although contamination of

samples with a high drug concentration from the venous blood draining the administration site is

considered unlikely, it is the most credible factor related to the experiment that would explain an

erroneously high bioavailability.

Bioavailability following extravascular administration can actually (rather than

erroneously) exceed 100% when the IV dose is not completely bioavailable. The main reason for

this to occur (other than technical issues) is metabolism by the lung, resulting in a first-pass

effect for IV administration similar to the hepatic first-pass effect observed for many drugs

following oral administration (Toutain & Bousquet-Melou 2004). First-pass lung uptake of

fentanyl and meperidine has been described in humans with the more lipophilic agent having

greater uptake (Roerig et al. 1987). Buprenorphine and fentanyl are both basic amines with high

lipophilicity, although buprenorphine is even more lipophilic (Avdeef et al. 1996). Hepatic and

intestinal extractions of morphine, naloxone, and buprenorphine in rats were compared, with

buprenorphine having the greatest percentage extraction and conjugation (Mistry & Houston

1987). Lastly, methadone, another basic amine and opioid has been reported to undergo

12

pulmonary metabolism in rats and rabbits (Wilson et al. 1976; Roerig et al. 1982). Altogether,

this suggests that pulmonary uptake and metabolism of buprenorphine is possible, but it has not

been documented in the literature. The relationship between plasma buprenorphine concentration

and analgesia is not direct, mainly influenced by slow equilibrium in the biophase (Doodnaught

et al. 2017), and the plasma buprenorphine concentration for adequate antinociception is not

fully elucidated and variable in dogs. Postoperative analgesia following ovariohysterectomy

utilizing a high-concentration formulation of buprenorphine administered IV, IM or SC at 0.02

mg kg−1 in conjunction with carprofen has been reported in dogs (Steagall et al. 2020). A greater

number of dogs required rescue analgesia after SC administration compared with IV, but the

plasma concentration at which this was required was lower in the SC group (0.39 ± 0.17 ng mL−1

versus 1.60 ± 0.38 ng mL−1). A separate study compared SC buprenorphine (0.02 mg kg−1) with

sustained-release SC buprenorphine (0.2 mg kg−1) (Nunamaker et al. 2014). Only two out of 20

dogs had breakthrough pain with plasma buprenorphine concentrations of 2.26 and 2.08 ng

mL−1; however, meloxicam was administered preoperatively to all dogs. A third study of dogs

undergoing ovariohysterectomy compared low (0.02 mg kg−1) and high (0.12 mg kg−1) dosages

of oral transmucosal versus IV buprenorphine (Ko et al. 2011). The results indicated that seven

out of nine dogs with buprenorphine plasma concentrations ≤ 0.6 ng mL−1 required rescue

analgesia, but the range for buprenorphine plasma concentration requiring rescue analgesia

extended to 5.41 ng mL−1.

Prior thermal threshold research in cats has led to similar values for various routes of

administration, with a buprenorphine EC50 (effective concentration in 50% of animals) of 2.13

ng mL−1 and “analgesia offset” of 2.3 ± 2.0 ng mL−1, described by Doodnaught et al. (2017), and

Taylor et al. (2016), respectively. Additionally, an EC50 of 1.83 ng mL−1 has been published

13

when comparing IV and oral transmucosal administration in cats, but there was a total range of

1.26–8.26 ng mL−1 across both treatments (Robertson et al. 2005). When comparing the different

species, routes of administration and dosages, there is not a clear cut-off for a buprenorphine

plasma concentration that would be expected to provide antinociception after 0.12 mg kg−1 SC,

although all dogs in the present study had a plasma concentration ≤ 2 ng mL−1 at 24 hours.

Pharmacodynamic testing is necessary to determine if the plasma concentration present at 24

hours provides analgesia.

The reported volume of distribution at steady state (Vss) is 9,685 mL kg−1 in the present

study, indicating a large distribution into tissue consistent with an agent that is highly lipophilic

and is higher than in other studies. However, reported Vss for buprenorphine have varied

immensely, such as 4590 ± 600 mL kg−1 following Simbadol (0.02 mg kg-1) IV in dogs (Steagall

et al. 2020). Reports of Vss after other formulations of buprenorphine (0.02 mg kg−1)

administered IV include 424.3 ± 19.8 mL kg−1 (Pieper et al. 2011), 4680.1 mL kg−1 (Barletta et

al. 2018) and 9500 ± 1900 mL kg−1 (Abbo et al. 2008).

The dosage chosen for the present study was higher than the average clinical dosage of

0.02 mg kg−1, but lower than the label dosage of Simbadol for cats, 0.24 mg kg−1 (Zoetis 2017,

https://www.zoetisus.com/products/cats/simbadol/pdf/simbadol-pi.pdf). The label SC dosage of

Simbadol has been compared with Simbadol (0.12 mg kg−1) administered IV and oral

transmucosally, with SC Simbadol achieving a longer duration of antinociception (Doodnaught

et al. 2017). An earlier study in cats compared various dosages of several concentrations of

buprenorphine, all administered SC (Taylor et al. 2016). The results of that study determined that

buprenorphine (0.12 mg kg−1) provided antinociception for at least 24 hours without significant

side effects, regardless of concentration or formulation of the buprenorphine.

14

A limitation of the present study is that only male Beagle dogs were utilized. There is no

indication of differences in pharmacokinetics between sexes for buprenorphine in cats and dogs;

however, most published studies have not had sufficient power to conclude a lack of difference

(Steagall et al. 2013; Taylor et al. 2016; Doodnaught et al. 2017; Barletta et al. 2018). An

additional limitation is that pharmacodynamic testing was not performed as the present study

was oriented at description of the pharmacokinetic parameters; clinical antinociception from the

dosing cannot be directly correlated.

15

V. CONCLUSION

The high-concentration formulation of buprenorphine evaluated in the present study

showed a rapid absorption phase, followed by a delayed slow absorption phase, when

administered SC in male Beagle dogs. Sedation, whining, hyperptyalism and reduction in

appetite were seen following IV and SC administration, although the presence of each varied

highly among dogs. IV catheter placement and sampling was tolerated well by all dogs for both

treatments. Further study is indicated to determine if the obtained buprenorphine plasma

concentrations correlate with clinical antinociception.

16

VI. FIGURES 1 AND 2

Figure 1 Mean ± standard deviation buprenorphine plasma concentrations in six male Beagle

dogs following intravenous (IV; circles) and subcutaneous (SC; squares) administration of high-

concentration buprenorphine (0.12 mg kg−1; 1.8 mg mL−1).

17

Figure 2 Individual observations (symbols) and population predictions (solid line) in

buprenorphine plasma concentration in six male Beagle dogs following (A) intravenous and (B)

subcutaneous administration of high-concentration buprenorphine (0.12 mg kg−1; 1.8 mg mL−1).

18

VII. TABLE 1

Pharmacokinetic parameters, typical value, coefficient of variation and interindividual variability

(%) in six male Beagle dogs following intravenous and subcutaneous administration of high-

concentration buprenorphine (0.12 mg kg−1; 1.8 mg mL−1). The coefficients of variation were

obtained by bootstrapping.

Parameter Estimate Coefficient of

variation

Interindividual

variability (%)

V1 (mL kg−1) 900 17 33

V2 (mL kg−1) 2425 9 NE

V3 (mL kg−1) 6360 17 28

CL (mL minute−1 kg−1) 25.7 10 21

CL2 (mL minute−1 kg−1) 107.5 37 74

CL3 (mL minute−1 kg−1) 5.7 27 61

tlag1 (minute)

tlag2 (minutes)

0.72

226

41 103

24 42

ka1 (minute−1)

ka2 (minute−1)

0.078

0.002

32 63

18 36

F (%)

Fract (%)

131

63

15 40

57 85

C(0) (ng mL−1) 133 Na na

tmax (minutes) 12 Na na

Cmax (ng mL−1) 30.3 Na na

8.9 Na na

19

ka1 t½ (minutes)

ka2 t½ (minutes)

347 Na na

Vss (mL kg−1) 9,685 Na na

t½ α (minutes) 3.6 Na na

t½ β (minutes) 84 Na na

t½ γ (minutes) 963 Na na

C(0): concentration at time 0 following bolus intravenous administration; CL: metabolic

clearance; CL2: first distribution clearance; CL3: second distribution clearance; Cmax: peak

plasma concentration following subcutaneous administration; F: bioavailability following

subcutaneous administration; Fract: fraction of the bioavailable dose absorbed during the rapid

absorption phase; ka1: rate constant for the rapid absorption phase following subcutaneous

administration; ka2: rate constant for the slow absorption phase following subcutaneous

administration; ka1 t½: half-life of the rapid absorption phase following subcutaneous

administration; ka2 t½: half-life of slow absorption phase following subcutaneous administration;

na: not applicable because the estimate is calculated from parameters estimated by the model;

NE: not estimated because of insufficient data as assessed by an η shrinkage >0.4 (see Appendix

SA for details); t½ α: half-life of the fast distribution phase; t½ β: half-life of the slow distribution

phase; t½ γ: terminal half-life; tlag1: delay of the rapid absorption phase following subcutaneous

administration; tlag2: delay of the slow absorption phase following subcutaneous administration;

tmax: time to peak plasma concentration following subcutaneous administration; V1: volume of

the central compartment; V2: volume of the first peripheral compartment; V3: volume of the

second peripheral compartment; Vss: volume of distribution at steady state.

20

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24

IX. APPENDIX SA

Pharmacokinetic modeling:

The final model estimated volumes (V), clearances (CL), bioavailability (F), absorption

rate constants (ka), absorption delays (tlag), and fraction of the bioavailable dose absorbed

during the rapid absorption phase (Fract). The value of Fract was constrained between 0 and 1

through the use of a logit function. The fraction of the bioavailable dose absorbed by the slow

absorption phase was assumed to be 1-Fract. Different error and covariance structures were

tested; a multiplicative error was used in the final model. A diagonal covariance structure (diag)

was used for some random effects, and correlation between random effects was calculated for

other ones (block). Typical values (tv) of the parameters and random effects (η) to account for

interindividual variability were estimated by the model. For each estimate, the parameter in the

individual i was estimated as 𝑃𝑖 = 𝑡𝑣𝑃𝑖 × 𝑒𝜂𝑖, with a different η for each P. The random effects η

were assumed to be normally distributed and to have a mean of 0 and a variance of ω2. The

interindividual variability was calculated for each parameter estimated by the model as

√𝑒𝜔2− 1 × 100, and corresponds to a coefficient of variation (Mould & Upton 2013). Random

effects were removed from the final model if excessive shrinkage (>0.4) was present, indicating

that the data were insufficient to estimate between-subject variability for the parameter(s)

concerned.

Reference

Mould DR, Upton RN (2013) Basic concepts in population modeling, simulation, and model-

based drug development-part 2: introduction to pharmacokinetic modeling methods. CPT

Pharmacometrics Syst Pharmacol 2, e38.

Model code

test(){

deriv(SC1 = - Ka1 * SC1)

deriv(SC2 = - Ka2 * SC2)

deriv(A1 = - Cl * C + (Ka1 * SC1) + (Ka2 * SC2) - Cl2 * (C - C2) - Cl3 * (C - C3))

deriv(A2 = Cl2 * (C - C2))

25

deriv(A3 = Cl3 * (C - C3))

dosepoint(A1)

dosepoint(SC1, tlag = (Tlag1), bioavail = (F * ilogit(Fract)))

dosepoint(SC2, tlag = (Tlag2), bioavail = (F * (1-ilogit(Fract))))

C = A1 / V

C2 = A2 / V2

C3 = A3 / V3

error(CEps = 0.302894684125625)

observe(CObs = C * (1 + CEps))

stparm(V = tvV * exp(nV))

stparm(V2 = tvV2)

stparm(V3 = tvV3 * exp(nV3))

stparm(Cl = tvCl * exp(nCl))

stparm(Cl2 = tvCl2 * exp(nCl2))

stparm(Cl3 = tvCl3 * exp(nCl3))

stparm(Tlag1 = tvTlag1 * exp(nTlag1))

stparm(Tlag2 = tvTlag2 *exp(nTlag2))

stparm(Ka1 = tvKa1 * exp(nKa1))

stparm(Ka2 = tvKa2 *exp(nKa2))

stparm(F = tvF * exp(nF))

stparm(Fract = tvFract + nFract)

fixef(tvV = c(, 941.340479006028, ))

fixef(tvV2 = c(, 2419.21936507735, ))

fixef(tvV3 = c(, 6169.61075172764, ))

fixef(tvCl = c(, 25.7371871404421, ))

fixef(tvCl2 = c(, 101.085407531662, ))

fixef(tvCl3 = c(, 5.54512155819806, ))

fixef(tvTlag1 = c(0, 0.749681722695445, ))

fixef(tvTlag2 = c(, 266.486690968933, ))

fixef(tvKa1 = c(, 0.0830340168948763, ))

26

fixef(tvKa2 = c(, 0.00170709222828607, ))

fixef(tvF = c(, 1.31889783829645, ))

fixef(tvFract = c(, 0.466791591974438, ))

ranef(diag(nV3, nCl2, nTlag1, nTlag2, nFract) = c(0.079749682, 0.35354777,

0.7754824, 0.14405324, 0.51763957))

ranef(block(nV, nCl, nCl3, nKa1, nKa2, nF) = c(0.11499939, 0, 0.03663256, 0, 0,

0.25198087, 0, 0, 0, 0.44715331,0, 0, 0, 0, 0.08923388, 0, 0, 0, 0, 0, 0.1194417))

}

Goodness-of-fit plots (Supplementary Figures 1-4)

Supplementary Figure 1 Conditional weighted residuals (CWRES) versus time (IVAR)

27

Supplementary Figure 2 Conditional weighted residuals (CWRES) versus population predicted

values (PRED)

Supplementary Figure 3 Observed concentrations (DV) versus individual predicted values

(IPRED)

28

Supplementary Figure 4 Observed concentrations (DV) versus population predicted values

(PRED)