Materials and Methods

Animals—Six Dorset cross sheep (5 castrated males and 1 female) with a mean ± SD age of 2.2 ± 0.03 years were included in the study. The sheep had been obtained from the University of Pennsylvania Comparative Orthopedic Research Laboratory. All were healthy as determined by physical examination. The Institutional Animal Care and Use Committee at the University of Pennsylvania approved this study protocol.

One week before the pharmacokinetic trials began, the sheep were acclimatized to group housing in a stall (3.61 × 3.45 m) with concrete flooring and straw bedding that was replaced daily. Free access to timothy grass (Phleum pretense), orchard grass hay (Dactylis glomerata), and water was provided.

Study design—A randomized crossover design with a 10-day washout period was used. Twelve hours before pharmacokinetic evaluation began, sheep were weighed to determine drug doses and IV catheters were placed. Each had a jugular catheter placed for the purpose of blood sample collection. An additional jugular catheter dedicated to drug administration was placed in sheep that were to receive meloxicam IV.

In the first phase of the study, 3 randomly selected sheep received an IV injection of meloxicam^a at a dose of 0.5 mg/kg. The dose volume was rounded to the nearest tenth that could be accurately measured by the calibrated gradations of a 6-mL syringe. After the meloxicam injection, the catheter used was flushed with 10 mL of saline (0.9% NaCl) solution and then immediately removed. The remaining 3 sheep were assigned to receive meloxicam^b PO at 1.0 mg/kg. The dose was rounded to the nearest whole tablet, resulting in an actual mean dose of 0.99 mg/kg (range, 0.87 to 1.11 mg/kg). For oral administration, tablets were dissolved in 60 mL of water and administered via an ororuminal tube, immediately after which the tube was flushed with 1 L of water. A 10-day washout period was allowed, which reportedly is adequate to ensure clearance of the drug from cattle and goats.^9,11 Afterward, phase 2 of the study began, with sheep that had undergone meloxicam administration PO receiving the drug IV and those that had undergone IV administration receiving meloxicam PO.

Blood sample collection—An animal handler restrained each sheep, and blood samples were collected via the dedicated jugular catheter at 0.05, 0.1, 0.17, 0.33, 0.67, 1, 3, 6, 12, 24, 48, 72, and 96 hours after IV meloxicam administration or at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, and 96 hours following PO administration. Catheter patency was maintained after each sample was collected by flushing with 2 mL of saline solution containing 3 U of heparin sodium^c/mL. Collected samples were immediately transferred to an additive-free blood collection tube and stored on ice before processing by centrifugation for 10 minutes at 1,500 × g. Harvested serum was transferred to vials^d and frozen at −70°C until analysis. All samples were analyzed within 60 days after collection.

Serum meloxicam concentration measurement—Serum concentrations of meloxicam were measured via high-pressure liquid chromatography^e with mass spectrometry.^f Serum samples or serum standards (0.1 mL) were added to 0.4 mL of a solution containing methanol, 0.1% formic acid, and the internal standard piroxicam (100 ng/mL). The samples were mixed with a vortex device for 5 seconds and centrifuged for 10 minutes at 10,000 × g to obtain a precipitate. The supernatant (200 μL) was transferred to an injection vial with the injection volume set to 10 μL. The mobile phase consisted of acetonitrile and formic acid (0.1%) at a flow rate of 0.4 mL/min. The concentration of this mobile phase began at 85% formic acid (0.1%) from 0 to 0.5 minutes with a linear gradient to 50% formic acid (0.1%) at 2.5 minutes, which was maintained until 3 minutes, followed by a linear gradient to 85% formic acid (0.1%) at 4 minutes with a total run time of 5 minutes. Separation was achieved with a C₈ column^g maintained at 40°C.

The standard curve of meloxicam concentration determined through use of sheep serum was linear from 0.025 to 5.0 μg/mL and was accepted when the correlation coefficient exceeded 0.99 and measured values were within 15% of the actual values. The lower limit of quantification, defined as the lowest concentration on a linear standard curve with predicted concentrations within 15% of the actual concentration, was 0.025 μg/mL. The qualifying ions (m/z) for meloxicam and piroxicam were 352.09 and 332.12, respectively. The quantifying ions for meloxicam and piroxicam were 114.90 and 95.10, respectively. Intraday accuracy of the assay for meloxicam in sheep serum was 99 ± 3% of the actual concentration, whereas the intraday coefficient of variation was 2%, as determined through replicates of 5 for each of the following meloxicam concentrations: 0.05, 0.50, and 5.0 μg/mL.

Pharmacokinetic analysis—Pharmacokinetic analyses were performed with computer software^h by use of noncompartmental methods. The parameters included the AUC from time 0 to infinity as determined via the linear trapezoidal rule, percentage of the AUC extrapolated to infinity, serum clearance, serum clearance per bioavailability, concentration extrapolated to time 0 via log-linear of the first 2 time points following IV administration, apparent volume of distribution in a steady state, Vz, Vz/F, Λz, T_1/2Λz, and MRT. The Cmax and Tmax were determined directly from the data gathered after oral meloxicam administration. The extrapolated portion of the AUC was determined by multiplying the last measured serum concentration by the Λz. The range of the Λz was determined by visual inspection of the serum profile and determined by linear regression of time and natural logarithm (ln) of the serum concentration. The Vz was determined by dividing the dose by Λz•AUC. The apparent volume of distribution in a steady state was determined by multiplying the MRT by the clearance. The clearance was determined by dividing the dose by the AUC. Bioavailability was estimated by dividing the AUC per dose for PO administration by the AUC per dose for IV administration for each sheep. The MAT was estimated by subtracting the MRT for IV administration from the MRT for PO administration.

Statistical analysis—Pharmacokinetic values are reported as geometric means, medians, and ranges because the distribution was presumed to be log-normal. No statistical tests were performed.

Results

Animals—The mean ± SD body weight of the sheep during the first and second study phases was 55.3 ± 3.7 kg and 55.6 ± 4.4 kg, respectively. No adverse effects were observed following IV or PO administration of meloxicam.

Pharmacokinetics—Mean serum concentrations versus time for meloxicam administered IV at 0.5 mg/kg or PO at a mean dose of 0.99 mg/kg (range, 0.87 to 1.11 mg/kg) were graphically displayed (Figure 1). The geometric mean T_1/2Λz for IV administration was 14.0 hours (range, 10.5 to 17.0 hours) resulting from a geometric mean Vz of 0.204 L/kg (range, 0.171 to 0.272 L/kg) and a geometric mean clearance of 0.17 mL/min/kg (range, 0.12 to 0.27 mL/min/kg; Table 1). One male sheep was excluded from analysis of the IV administration data because of an apparent extravascular administration of meloxicam, which was evident by a pharmacokinetic profile with an absorptive component.

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Mean ± SD serum concentrations of meloxicam at various points after IV (actual dose, 0.5 mg/kg; black circles) and PO (mean dose, 0.99 mg/kg; range, 0.87 to 1.11 mg/kg; white circles) administration of the drug to 6 healthy sheep.

Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.779

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The geometric mean Cmax, Tmax, and T_1/2Λz for meloxicam administered PO were 1.72 μg/mL (range, 1.45 to 1.93 μg/mL), 19.0 hours (range, 12.0 to 24.0 hours), and 15.4 hours (range, 13.2 to 17.7 hours), respectively (Table 2). Mean bioavailability and MAT were 72% (range, 40% to 125%) and 12.4 hours (range, 9.1 to 17.0 hours), respectively. Because data from 1 sheep were excluded from analysis because of extravascular administration, bioavailability and MAT were calculated from the IV administration data from the remaining 5 sheep.

Discussion

The present study was conducted to determine the pharmacokinetics of meloxicam when administered IV and PO, including bioavailability. The dose of meloxicam used for each administration route was selected from published information regarding other domestic ruminants. Pharmacokinetic data have been reported for meloxicam administered IV at a uniform dose of 0.5 mg/kg in sheep, goats, and cattle.^9,11,15 The dose of meloxicam administered PO to the study sheep (1.0 mg/kg) was determined from findings of an analgesic study¹⁶ in which lameness was induced in sheep with turpentine. This higher dose was used as a result of a poor analgesic response observed when meloxicam was administered PO at 0.5 mg/kg in lambs following mulesing in a different study.¹⁷ No pharmacokinetic values were determined in either study; however, values were determined in a study⁹ in cattle that received meloxicam administered PO at 1.0 mg/kg. Although no adverse effects were observed in the sheep in our study after receiving meloxicam PO at 1.0 mg/kg, toxicity studies must be performed before recommendations for this use can be made. Furthermore, efficacy studies are needed to determine the dose of meloxicam needed to achieve analgesia when administered PO.

The dose of meloxicam differed between IV and PO administration in the present study. Although dose-dependent pharmacokinetics cannot be excluded, in other species including horses and humans, absorption of meloxicam is considered independent of dose with linear plasma meloxicam concentrations.^7,18 Additionally, despite administration of a higher dose PO, the peak serum concentrations achieved were still less than those achieved after IV administration and the serum concentrations on the terminal portions of the curves were similar for IV and PO administration. Given the linearity of meloxicam concentrations observed in other species and in our study, use of different doses for the determined pharmacokinetic parameters in sheep was justified.

Our findings suggested meloxicam has a high mean oral bioavailability (72%) in sheep. Interestingly, this mean value was similar to that reported for goats (79%)⁹ but lower than that reported for cattle (100%).¹¹ The Cmax was achieved after 19 hours in the study sheep. Although the Tmax was longer in sheep than has been reported for cattle (11.6 hours)¹¹ and goats (15.0 hours),⁹ the Cmax in sheep (1.72 μg/mL) was less than in cattle (3.10 μg/mL)¹¹ and greater than in goats (0.736 μg/mL).⁹ The delayed MAT (12.4 hours) suggested that meloxicam absorption in sheep occurred mostly distal to the 4-compartment gastric tract, with primarily intestinal absorption; it also suggested drug storage within the rumen. This finding contrasts with meloxicam absorption in monogastric animals such as rats and horses, in which absorption is relatively rapid, occurring over a long section of the gastrointestinal tract.^7,19

The delay in absorption observed in the present study could be a result of the feeding protocol used. In horses, feeding protocols can influence the absorption of NSAIDs administered PO.^21,22 In vitro and in vivo studies²² have shown that absorption of NSAIDs including phenylbutazone and flunixin meglumine when administered PO is delayed by binding of drugs to hay and digesta. Delayed absorption also occurs in humans and horses in a fed state.^7,18 However, in horses, bioavailability of these NSAIDs is not affected by the availability of feed.^7,21 It is unknown whether meloxicam binds to hay or digesta in ruminants, influencing bioavailability. However, because most animals will not have had food withheld in clinical settings, the results for the study sheep may reflect the pharmacokinetics of orally administered meloxicam in a typical clinical setting. Sheep were fed free-choice timothy grass and orchard grass hay only, which differs from feeding protocols in other ruminant studies,^9,11 which included feeding concentrates. In addition to the potential of meloxicam to bind to hay, NSAIDs are lipid-soluble, weak organic acids and pH can affect absorption,²³ which in turn can affect pharmacokinetic values.

A mean T_1/2Λz of 15.4 hours for meloxicam administered PO at 1.0 mg/kg was achieved in the study sheep. This terminal half-life value is less than that of meloxicam administered PO to cattle at 1.0 mg/kg, PO (27.5 hours).⁹ In contrast, the sheep had a longer terminal half-life than did goats that received meloxicam at 0.5 mg/kg, PO (11.8 hours).¹¹ Interspecies differences, data analysis techniques, or random variability may have contributed to these incongruent findings. Additionally, the longer mean T_1/2Λz of meloxicam when administered PO versus IV is consistently observed in other ruminants.^9,11

The terminal half-life of meloxicam in sheep was related to a restricted Vz (0.204 L/kg) and slow clearance (0.17 mL/min/kg). This restricted Vz has been found for NSAIDs in other species.^{9,11,14,15,18} Most likely, the restricted volume of distribution was due to extracellular drug distribution, a high ionization state at the sheep's physiologic pH, and the drug's high plasma protein binding ability.^15,18

The speed of meloxicam clearance varies among species. Evidence of recirculation of the drug within the intestinal lumen of humans has been suggested as a reason for slower elimination.¹⁸ Meloxicam clearance in the study sheep was faster than that reported for cattle (0.10 mL/min/kg)¹¹ and slower than that reported for goats (0.30 mL/min/kg).⁹ In comparison, the elimination kinetics in ruminants are consistent with those reported for NSAIDs. The greater elimination values in goats versus sheep are consistent with those of previous comparisons.¹⁵ Additionally, the lower elimination values in cattle are consistent with those for other NSAIDs such as flunixin meglumine and phenylbutazone.^23,24 In humans, meloxicam is primarily metabolized in the liver largely through cytochrome P450 2C9, with equal renal and fecal elimination.¹⁸ Attributable activity and concentration differences in these biotransformation enzymes in the liver, specifically cytochrome P450 2C9, have been identified in sheep, cattle, and goats.²⁵

Although analgesic effects are not evaluated in pharmacokinetic studies, a previous study¹⁶ showed that meloxicam has antinociceptive effects in sheep. Meloxicam administered once at 1.0 mg/kg, IV, provided some alleviation of signs of pain in sheep with experimentally induced lameness.²⁴ Additionally, favorable pharmacokinetics of orally administered meloxicam in sheep include a longer half-life than with other NSAIDs such as flunixin meglumine.²⁴ The pharmacokinetic profile of meloxicam in the present study suggested that oral administration should occur 24 hours prior to noxious stimulus exposure so that peak drug concentrations will coincide with the onset of tissue damage. However, studies of the pharmacokinetic-pharmacodynamic profiles of orally administered meloxicam in sheep would be necessary to determine the appropriate dosing regimen to treat acute or chronic pain states. In addition to analgesic efficacy studies, therapeutic safety indices are necessary prior to clinical application.

In the United States, meloxicam administration in sheep would constitute extralabel drug use. Because of the lack of analgesics approved for use in food animals within the United States, extralabel drug use is permitted under AMDUCA provided that specific guidelines are met.²⁶ However, as a stipulation of AMDUCA, no volatile tissue residues can be detectable in animals or animal products used for human consumption. In several European countries, meloxicam is approved for use in cattle as an adjunctive treatment for acute respiratory disease, acute mastitis, and diarrhea when administered IV or SC at 0.5 mg/kg with a withdrawal period of 15 days for meat and 5 days for milk.²⁷ The duration of drug withholding from animals used for meat can be estimated by calculating 10 half-lives, which under linear elimination kinetics would indicate elimination of 99.9% of drug residues from tissues.²⁸ Given the maximum half-life observed in the present study and with rounding to the nearest whole day, a meloxicam withdrawal period of 8 days would be calculated by this method. However, in the absence of associated tissue residue studies in sheep, for a 1-time administration, the authors recommend a conservative drug withholding period of 21 days on the basis of the available information in cattle.ⁱ Amounts and durations of residues will change with 1-time versus multiple doses and should be considered when determining an appropriate withholding period.