Materials and Methods

Eight adult White Leghorn hens were obtained from a commercial source and housed in a climate-controlled facility for 2 weeks prior to undergoing any procedures. Hens were determined to be healthy on the basis of physical examination findings. The birds were housed individually in large wire cages (61 × 122 × 91.5 cm) that were adjacent to each other so that each hen could see all other hens. Birds were provided water, layer pellets, and scratch feed ad libitum. Fresh greens were provided daily, and mealworms were provided intermittently as enrichment. The birds were maintained on a cycle of 14 hours of light and 8 hours of darkness to encourage egg laying. All study procedures were approved by the University of Tennessee Institutional Animal Care and Use Committee.

At the start of the study, hens were weighed, and a dose of 1 mg of meloxicam^a/kg was calculated. A blood sample (0.3 mL) was collected from each hen on day 0 immediately before administration of the drug (0 hours) via syringe into the oral cavity distal to the glottis. A blood sample (0.3 mL) was then collected at 10, 20, and 30 minutes and 1, 2, 4, 8, 12, 24, and 48 hours after dose administration. Blood samples were collected from a jugular or basilic vein with an insulin syringe and 29-gauge needle. Eggs laid by the dosed hens were collected for 3 weeks starting on the day of meloxicam administration (day 0). Eggs were kept refrigerated until analyzed.

Collected blood samples were each placed into a lithium heparin plasma separator tube and placed on ice. Within 2 hours after collection, each blood sample was centrifuged for plasma separation; the plasma sample was placed into a cryogenic vial and then kept frozen at −80°C until analysis. Plasma samples were analyzed by the University of Tennessee Pharmacology Laboratory within 2 days after collection, and egg samples were analyzed within 2 weeks after collection.

Analysis of plasma and egg white (albumen) samples was conducted with a method developed by Cox et al.⁶ Briefly, analysis of meloxicam in plasma and egg white samples was performed by reversed-phase HPLC. The system consisted of a separations module, a UV absorbance detector, and a computer equipped with analytic software.^b The compounds were separated on a column^c (4.6 × 250 mm; particle size, 5 μm) with a 5-μm guard column.^c The mobile phase was a mixture of 10 mL of glacial acetic acid in 1 L of H₂O (pH 3.0 [adjusted with sodium hydroxide]; 50%) and acetonitrile (50%). Absorbance was measured at 360 nm with a flow rate of 1 mL/min.

Standard curves for egg white analysis were prepared by means of fortifying untreated egg whites with meloxicam to produce a linear concentration range of 5 to 1,500 ng/mL. Calibration samples were prepared exactly as were the egg white samples. The lower limit of quantification during validation was 5 ng/mL The intra-and interassay variability ranged from 0.5% to 6.6%, and the calculated mean recovery for meloxicam was 90%. Standard curves for plasma analysis were prepared by means of fortifying samples of untreated plasma with meloxicam to produce a linear concentration range of 5 to 10,000 ng/mL. Calibration samples were prepared exactly as were the plasma samples. The lower limit of quantification during validation was 5 ng/mL. The intra- and interassay variability ranged from 1.1% to 10%, and the calculated mean recovery for meloxicam was 95%.

Meloxicam was extracted from egg yolks by means of a solid-phase extraction technique using HPLC. For each egg, 1 mL of yolk was placed in a tube, followed by 75 μL of the internal standard (piroxicam, 5 μg/mL) and 200 μL of 1M HCl and 2 mL of acetonitrile. Samples were vortexed and centrifuged, and then the supernatant was removed and evaporated. The residue was redissolved in methanol and water (1:9 ratio) and added to an extraction cartridge.^d Elution was performed with 2 mL of acetonitrile and methanol (vol/vol, 90:10); the eluate was then evaporated with nitrogen and redissolved in the mobile phase. Standard curves for egg yolk analysis were prepared by means of fortifying untreated egg yolks with meloxicam to produce a linear concentration range of 5 to 1,500 ng/mL. The lower limit of quantification during validation was 5 ng/mL, and the limit of detection was 2.5 ng/mL. Intra- and interassay variability ranged from 1.8% to 7.8%, and the calculated mean recovery for meloxicam was 85%.

Pharmacokinetic parameters for meloxicam were calculated with computer software.^e Values for plasma half-life, maximum plasma concentration, time to maximum plasma concentration, and AUC_0-∞ of meloxicam were calculated from noncompartmental analysis. Area under the plasma concentration–time curve was calculated by use of the log-linear trapezoidal rule. Mean transient time from time 0 to infinity was calculated as the total area under the first movement curve divided by AUC_0–∞.

Results

Hens were observed daily for activity levels and food consumption, and no changes occurred during the 3-week study period. Plasma samples obtained from all 8 hens at 11 time points each were analyzed by HPLC. The mean ± SD terminal half-life, maximum concentration, and time to maximum concentration were 2.79 ± 1.01 hours, 7.21 ± 3.29 μg/mL, and 2.0 ± 0.92 hours, respectively (Table 1). A plasma meloxicam concentration–time curve was generated (Figure 1). Eggs were collected from hens daily for 3 weeks following meloxicam administration. Only 5 hens laid eggs consistently (every day or every other day) through the collection period. Hens did not lay eggs daily, and 1 hen laid only 1 egg during the collection period. No drug was detected in egg yolks or egg whites after 8 and 4 days, respectively. Eggs were collected for 3 weeks, but the HPLC analysis of egg white and yolk samples was discontinued for each hen once at least 2 consecutive eggs from that hen had no detectable meloxicam. Median meloxicam concentrations in egg yolks and egg whites for all hens for days 0 to 13 were summarized (Table 2).

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Mean plasma concentration of meloxicam over time following PO administration of a single 1-mg/kg dose to 8 domestic hens. A blood sample (0.3 mL) was collected immediately before (0 hours) and at intervals up to 48 hours after drug administration from each of the hens. Plasma samples were kept at −80°C and analyzed by reversed-phase HPLC within 2 days after collection.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.965

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Discussion

In the present study, the pharmacokinetics of meloxicam in domestic chickens following a single PO dose of 1 mg/kg was determined. Pharmacokinetic evaluations of meloxicam administered PO in other avian species have been recently reported.^7,8 Comparison of these data revealed that the half-life of meloxicam was shortest in the chickens of the present study (2.79 hours), compared with findings in Hispaniolan Amazon parrots (15.8 hours), red-tailed hawks (3.97 hours), and great horned owls (5.07 hours). The time to maximum plasma meloxicam concentration in the study chickens was 2.0 hours, which was shorter than that in parrots (5.0 hours) and owls (7.8 hours), but longer than that in hawks (0.73 hours). In the study chickens, the maximum plasma meloxicam concentration achieved was 7.21 μg/mL, whereas that in parrots was 3.7 μg/mL; both of these species were administered meloxicam PO at a dose of 1 mg/kg. The hawks and owls were administered meloxicam PO at a dose of 0.5 mg/kg and had comparatively lower maximum plasma meloxicam concentrations of 0.182 and 0.368 μg/mL, respectively. Although sample analysis method can affect some pharmacokinetic parameters, the variability of results among these avian species emphasizes the importance of species-specific study findings as a basis for clinical recommendations.

Although the pharmacokinetics of meloxicam in chickens has been studied,^2–5 a therapeutic plasma concentration has yet to be determined. In Hispaniolan Amazon parrots, it has been suggested that a mean plasma meloxicam concentration of 3.5 μg/mL should adequately provide analgesia⁷; on the basis of that finding, the same dose (1 mg/kg) was selected for use in the chickens of the present study. However, as for any drug, one should anticipate species differences in metabolism; paired pharmacokinetic and pharmacodynamic assessments in a given avian species are needed to determine species-specific recommendations for treatment with meloxicam.^7,8 The therapeutic plasma meloxicam concentrations reported for humans⁹ should also be considered when dosing recommendations are made. On the basis of a therapeutic plasma concentration in humans of 0.5 to 1.5 μg of meloxicam/mL, PO administration of meloxicam at a dose of 1 mg/kg to domestic chickens should maintain therapeutic plasma meloxicam concentrations for 12 hours.⁹ Because accumulation of meloxicam could occur with repeated administration of doses, additional investigation of the pharmacokinetics of multiple meloxicam doses is needed before recommendation of a multiple-dose regimen. If the therapeutic plasma meloxicam concentration in domestic chickens is determined to be greater than that in humans (ie, more similar to that suggested for Hispaniolan Amazon parrots), more frequent administration or higher doses of meloxicam may be required. However, considering the potential adverse effects associated with NSAID administration, a lower-dose regimen is recommended until paired pharmacokinetic and pharmacodynamic studies can be performed.

To our knowledge, the present study was the first to examine drug residues in eggs following PO administration of meloxicam to domestic chickens. The drug was present for a longer period in egg yolks than in egg whites, which is consistent with the formation of eggs.¹⁰ The white (albumen) is laid onto the yolk later in egg formation; therefore, once meloxicam is no longer present systemically in an egg-laying hen, drug concentration in the egg white quickly decreases. Drug residues could persist in eggs for longer periods owing to the duration of yolk (follicle) formation, but residues of drugs are reported to typically persist for < 2 weeks.¹⁰ Although the number of eggs collected and analyzed each day during the present study was small, the data suggested that a 2-week withdrawal time should be adequate to avoid meloxicam residue in eggs following a single PO 1-mg/kg dose to domestic hens. Additional research will be needed to determine withdrawal times for multiple-dose regimens.

With the increasing popularity of backyard poultry, additional investigations of meloxicam, as well as other commonly used medications, will be needed to provide accurate withdrawal times for eggs intended for consumption. The present study has provided an initial assessment of the use of meloxicam in domestic laying hens, but additional research is needed to determine the pharmacokinetics and egg residues associated with multiple-dose administration as well as to establish species-specific therapeutic plasma meloxicam concentrations through paired pharmacokinetic and pharmacodynamic studies in domestic hens and other birds.