5.1 Animals and housing
North Carolina State University Institutional Animal Care and Use Committee approved this study. All animals were acquired from the North Carolina State University Swine Education Unit and transferred to the North Carolina State University College of Veterinary Medicine, where they were housed individually in metabolism cages (72oF), with a 12∶12 light:dark cycle, fed LabDiet 5084 (LabDiet, St. Louis, MO, USA) twice a day and had access to freshwater ad libitum. A total of fourteen healthy, castrated, male Yorkshire/Landrace cross pigs (weighing 23.1-35.4 kg) were enrolled to receive either flunixin or meloxicam (n=7 per treatment group). There is no hypothesis testing involved in this pharmacokinetics study, and therefore a power analysis is not required for estimating sample size as previously described (31). This study excluded any animals with hernias, diarrhea, lameness or any other clinical signs of disease and inclusion criteria were no prior treatment with flunixin or meloxicam. Pigs appeared healthy on physical examination by lack of any clinical signs. During catheter placement and interstitial fluid probe placement, temperature, heart rate and respiratory rate were monitored, and no abnormalities were noted. Pigs were randomly assigned to metabolism cages at any given trial by individuals not involved in the study. There were three trials consisting of 4 pigs/trial with 2 pigs being treated with flunixin and 2 pigs being treated with meloxicam, and one trial with one pig treated with flunixin and other pig with meloxicam. This allowed us to account for the effect of litter. Investigators were not blinded to sample collection or sample analysis at any stage of the study.
5.2 Catheter and Interstitial Probe Placement
Prior to the start of the study, pigs were moved to individual metabolism cages and allowed 4 days of acclimation. After the adjustment period pigs were sedated using an intramuscular injection of a combination of Telazol® (50 mg/mL tiletamine HCl and 50 mg/mL zolazepam HCl), ketamine (100 mg/mL) and xylazine (100 mg/mL) at a concentration of 0.6 mL/kg body weight. Using sterile technique, an 18 Ga x 15 cm catheter (SA1815; Mila International, Inc., Florence, KY, USA) was inserted into the right jugular vein and sutured to the skin using 2-0 monofilament suture and an extension attached.
At the time of catheter placement, an ultrafiltration probe (Canine UF Probe, BASi systems, W. LaFayette, IN, USA) was placed subcutaneously along the epaxial muscles using a previously described technique (32). The interstitial probe allowed for continuous collection of interstitial fluid (ISF). Pigs were able to recover for 36-48 hours following the placement of instrumentation. During this recovery period, patency of the catheter was maintained by removing the heparin lock (100 mg/mL), flushing the catheter with saline and replacing the heparin lock every 12 hours.
5.3 Drug Administration and Sample Collection
Pigs were administered a single intramuscular dose of either 0.4 mg/kg meloxicam (Meloxicam solution for injection 5 mg/mL, Putney, Inc., Portland, ME, USA), the labeled dose for pigs in Europe, or 2.2 mg/kg flunixin meglumine (Banamine-S®, Merck Animal Health, Summit, NJ, USA), the labeled dose for pigs in the US. The injection location was the neck of the pigs in accordance to the label instructions. Blood samples (3 mL) were collected via the jugular catheter and transferred to lithium heparinized tubes at 0 (baseline), 0.08, 0.17, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, 132, 156 and 180 h post-administration of flunixin or meloxicam. Blood samples were centrifuged at 3500 x g and the plasma collected for analysis of total drug concentrations.
Interstitial fluid samples were collected via the preplaced collection probes at 0 (baseline), 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, 132, 156 and 180 h post-dose and weighed to determine the volume collected. At the end of the experiment, the ISF probe was removed and the tubing length measured. A lag time for the ISF collection was calculated to account for the time taken for the sample to travel along the ISF probe tubing. Interstitial fluid was used to quantify the free (protein unbound/pharmacologically active portion) drug concentrations in the tissues.
In order to determine drug concentrations in urine, animals were housed individually in metabolism cages to allow for collection of urine samples. Urine samples were collected at 0 (pretreatment), 4, 8, 12, 24, 48, 72, 96, 120, 144 and 168 h after drug administration. Urine was collected via a tray under the metabolism cages with a spout at the front of the cage and a stainless-steel bucket underneath to catch the urine and limit fecal contamination. Further details of the sample collection and calculation are described in our previous study in goats by Bublitz et al (2019) (2).. All plasma, ISF and urine samples were frozen at -80 ºC prior to analysis.
5.4 Tissue collection
After two consecutive negative (drug-free) urine samples (36-72 h for meloxicam, and 96-168 h for flunixin), each pig was euthanized. In order to minimize stress associated with euthanasia, the pigs were first sedated via intramuscular injection of 50:50 ketamine (100 mg/mL) and xylazine (100 mg/mL), equivalent to a final dose of 2.2 mg/kg ketamine and 2.2 mg/kg xylazine. After sedation, Euthasol® was administered through the jugular vein catheter at a dose equivalent to 85.9 mg/kg pentobarbital sodium and 11 mg/kg phenytoin sodium. Several biopsy punches were taken from each lobe of the liver and the entire left and right kidneys were taken from each pig in order to analyze tissues when the drug was no longer detectable in urine. These tissue samples were frozen at -20oC until analysis.
5.5 Drug Analysis
5.5.1 Plasma and Urine Sample Preparation
Flunixin and meloxicam plasma and urine samples were prepared using solid-phase extraction prior to UPLC-MS/MS analysis. Samples (300 µL) were pretreated with 300 µL of 4% phosphoric acid and vortexed for 10 seconds. Then, 500 µL of this pretreated sample was loaded onto an Oasis 1 mL 30 mg PRiME HLB cartridge (Waters Corp.), washed with 1 mL of methanol, and eluted from the cartridge with 500 µL of 90:10 (vol/vol) acetonitrile:methanol. The eluate was filtered through a 0.2μm PTFE Whatman Mini-UniPrep Syringeless Filter vial (GE Healthcare UK Limited., Buckinghamshire, UK) and then injected onto the UPLC-MS/MS system.
5.5.2 Tissue Sample Preparation
For all kidney and liver samples, a 0.1 g sub-sample was weighed into a 2‐mL bead mill tube containing 2.8‐mm ceramic beads (Fisher Scientific, Hampton, NH, USA). Then, 1 mL of acetonitrile was added to the tube and the contents homogenized 3 times for 15 seconds during each cycle at a speed of 5 m/s for kidney, and 4 m/s for liver, with a 10 second rest between cycles (BeadMill24, Fisher Scientific). Following homogenization, the tubes were centrifuged at 10,000 x g for 5 minutes. Then, 800 μL of supernatant was transferred to a 16 × 100 mm borosilicate glass tube containing 800 μL of acetonitrile and 400 μL of water. This mixture was vortexed gently for 10 seconds and then eluted through a 3 mL Captiva EMR-Lipid cartridge (Agilent Technologies, Inc., Santa Clara, CA, USA). The eluate was then evaporated to dryness at 55°C for 25 minutes. The sample was reconstituted in 300 μL of 1:1 acetonitrile:water, vortexed for 30 seconds, and the contents transferred a 0.2μm PTFE Whatman Mini-UniPrep Syringeless Filter vial and then injected onto the UPLC-MS/MS system.
5.5.3 UPLC-MS/MS Conditions
All samples were quantified by ultra-high-pressure liquid chromatography (UPLC) with mass spectrometric (MS/MS) detection (Waters Corp., Milford, MA, USA). The UPLC-MS/MS system consisted of a Xevo TQD tandem quadrupole mass spectrometer (Waters Corp.)
For flunixin samples, separation was achieved with a 2.1 mm x 100 mm, 1.7 μm Waters Acquity BEH Phenyl column (Waters Corp.). A gradient was used, and the initial mobile phase was 0.1% formic acid in water: 0.1% formic acid in acetonitrile (70:30 v/v) with a flow rate of 0.4 mL/min for the first 2.5 minutes. The mobile phase then switched to (10:90 v/v) from 2.5 min - 3.5 min. For the last 1.5 min of the run, the mobile phase was (70:30 v/v). The MS/MS was run in ESI+ mode. The quantification trace used was 297 → 279. Column temperature was 35 °C and sample temperature was ambient.
For meloxicam samples, separation was achieved with a 2.1 mm x 50 mm, 1.7 um Waters Acquity BEH C18 column (Waters Corp.) A gradient was used, and the initial mobile phase was 0.1% formic acid in water: 0.1% formic acid in acetonitrile (65:35 v/v) with a flow rate of 0.4 mL/min for the first minute. The mobile phase then switched to (10:90 v/v) from 1.0 min – 1.1 min. For the last 1.9 min of the run, the mobile phase was (65:35 v/v). The MS/MS was run in ESI+ mode. The quantification trace used was 352.043 → 115. Column temperature was 35 °C and sample temperature was 10 °C.
Validation standards were prepared over a linear range for each matrix (plasma, urine, kidney and liver) and were used to construct calibration curves. For the inter-day accuracy and precision, standards were repeated over 3 days. The concentrations analyzed varied by drug and by matrix and are shown in Table 3a below.
Drug
|
Matrix
|
# of concentrations
|
Concentrations (μg/mL)
|
Flunixin
|
Plasma
|
6
|
0.0005, 0.001, 0.01, 0.1, 0.5, 1
|
Urine
|
7
|
0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5
|
ISF
|
6
|
0.0005, 0.001, 0.002, 0.005, 0.01, 0.05
|
Liver
|
6
|
0.0001, 0.0005, 0.001, 0.01, 0.05, 0.1
|
Kidney
|
5
|
0.0005, 0.001, 0.01, 0.05, 0.1
|
Meloxicam
|
Plasma
|
7
|
0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1
|
Urine
|
6
|
0.005, 0.01, 0.05, 0.1, 0.5, 1
|
ISF
|
6
|
0.0005, 0.002, 0.005, 0.01, 0.025, 0.05
|
Liver
|
5
|
0.005, 0.01, 0.1, 0.5, 1
|
Kidney
|
6
|
0.005, 0.01, 0.05, 0.1, 0.5, 1
|
Table 3a: Concentrations and replicates used for the assay validation.
All calibration curves were linear with a R2 value of 0.99 or higher. Limit of quantification, inter-day accuracy and inter-day precision are presented in Table 3b for each analytical method.
Sample Analysis Parameters
|
Drug
|
Tissue
|
LOQ
|
Accuracy (%)
|
Precision (%)
|
µg/mL or µg/g
|
Mean
|
±SD
|
Mean
|
±SD
|
Flunixin
|
Plasma
|
0.0005
|
107
|
(6)
|
5
|
(3)
|
ISF
|
0.0005
|
99
|
(7)
|
6
|
(5)
|
Urine
|
0.0005
|
103
|
(5)
|
6
|
(3)
|
Liver
|
0.0001
|
100
|
(5)
|
5
|
(2)
|
Kidney
|
0.0001
|
100
|
(7)
|
8
|
(5)
|
Meloxicam
|
Plasma
|
0.001
|
100
|
(5)
|
6
|
(5)
|
ISF
|
0.001
|
97
|
(6)
|
6
|
(2)
|
Urine
|
0.001
|
104
|
(5)
|
4
|
(2)
|
Liver
|
0.005
|
100
|
(5)
|
7
|
(2)
|
Kidney
|
0.005
|
100
|
(4)
|
8
|
(2)
|
Table 3b: Limit of quantification (LOQ; μg/mL for fluids or μg/g for tissues), inter-day accuracy (%) and inter-day precision (%) for analytical methods. SD; standard deviation
5.6 Pharmacokinetic analysis
A noncompartmental analysis of drug plasma concentration vs. time profiles was performed with Phoenix WinNonLin software (version 8.0; Certara, Princeton, NJ, USA). The area under the plasma concentration–time curve from time zero to infinity (AUC0→∞; h*μg/mL) was calculated by linear log trapezoid method. The AUC0→∞ was used to calculate clearance per fraction absorbed (Cl/F; L/h/kg) and half‐life (T1/2; h). The volume of distribution (per fraction absorbed) (Vd/F; L/kg) was also calculated. Peak concentration (Cmax; μg/mL) and time at which maximum concentration occurs (Tmax; h) in plasma and urine was taken directly from the data from each pig.
Individual renal clearance values were estimated for each pig using the following equation: Renal Clearance (mL/h) = [(Ae/AUC)] (33), where Ae is the cumulative amount of drug excreted unchanged in the urine and AUC is the area under the plasma concentration-time curve to infinity. These values were then corrected for body weight for comparison with the total body clearance and reported as mL/h/kg in the Table 2.