Materials and Methods

Animals—Twenty-one (12 male and 9 female) green iguanas maintained in animal care and use facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care within the College of Veterinary Medicine were used in the study. All of the iguanas had been recently imported, were approximately 2 years old, weighed between 350 and 550 g, and had previously been maintained on an indigenous plant diet in outdoor enclosures in El Salvador. For the present study, iguanas were individually housed in ventilated plastic containers measuring 75 × 30 × 50 cm. Air temperatures ranged from 26° to 29°C during the day, and self-ballasted mercury vapor lamps^a positioned above 1 end of each container provided broad-spectrum lighting and surface basking areas of 37°C during the day. Relative humidity ranged from 60% to 90%, and photoperiod was set to a cycle of 12 hours of light and 12 hours of dark. Each iguana was provided with fresh water and mixed greens supplemented with calcium carbonate every morning. In addition, containers were liberally sprayed with water on a daily basis. Iguanas were allowed to acclimatize for 7 days prior to the start of the study, and all were eating, drinking, urinating, defecating, and behaving normally at the start of the study. Body weights were recorded at the start and end of the study.

Experimental design—The study protocol was approved by the University of Georgia's Institutional Animal Care and Use Committee. The study was conducted in 2 phases. Phase 1 consisted of a crossover pharmacokinetics study; 13 of the iguanas were administered a single dose (0.2 mg/kg) of meloxicam PO and, 14 days later, the same dose IV, and plasma meloxicam concentrations were measured. Phase 2 consisted of a toxicity study.

Pharmacokinetics study—For the pharmacokinetics study, food, but not water, was withheld for 24 hours prior to PO administration of meloxicam. Each iguana was physically examined and weighed; iguanas were individually numbered with a permanent marker pen on the cranium and a taped tag around the left pectoral limb. A baseline (time 0) blood sample (0.4 mL) was collected from the caudal (ventral tail) vein with a 1-mL syringe attached to a 23-gauge, 1-inch needle and placed into a plasma separator tube containing lithium heparin^b that was subsequently placed on ice. A 0.5-mL syringe containing meloxicam (0.2 mg/kg) suspension (1.5 mg/mL) for oral administration^c was attached to a 10-cm, ball-tipped, stainless steel feeding tube,^d and the feeding tube was advanced down the esophagus and into the stomach. The meloxicam was injected into the stomach, and 5 mL of sterile water was flushed through the feeding tube to ensure that all drug was administered. Additional blood samples were collected 3, 7, 12, 23, 47, and 72 hours after drug administration. All blood samples were kept on ice until centrifuged at 5,000 X g; all samples were centrifuged within 1 hour after collection. Plasma samples were placed in individual 0.5-mL microcentrifuge tubes^e and stored at –70°C until analyzed by means of high-performance liquid chromatography for meloxicam concentration.

After a 14-day washout period, the same 13 iguanas were given a single dose of meloxicam IV. Food was again withheld for 24 hours prior to drug administration. Each iguana was physically examined and weighed, and a baseline (time 0) blood sample (0.4 mL) was collected from the cranial region of the caudal vein with a 1-mL syringe attached to a 23-gauge, 1-inch needle. The syringe was carefully detached, leaving the needle in the vein, and the blood sample was placed in a plasma separator tube containing lithium heparin, which was placed on ice. A second 0.5-mL syringe containing meloxicam (0.2 mg/kg) suspension^f (0.04 mL/kg) was attached to the needle, and after aspiration of blood to ensure IV placement, the meloxicam was injected. For each iguana, the dose of meloxicam was calculated to take into account the volume in the needle hub. After the dose of meloxicam was administered, blood was again aspirated into the syringe and injected back into the iguana to ensure that all of the drug had been injected. The needle was withdrawn, and pressure was maintained on the injection site for 30 seconds. The iguana was then returned to its cage. Additional blood samples were collected from the caudal region of the caudal vein of each iguana 1, 4, 8, 12, 23, and 47 hours after IV administration. All collected blood samples were processed as described for samples collected after PO administration.

Toxicity study—Eight iguanas were used to study the potential toxicity of meloxicam. Two iguanas were given meloxicam at a dosage of 1 mg/kg, PO, every 24 hours for 12 days (10 times the daily dose approved for use in dogs), 2 iguanas were given meloxicam at a dosage of 5 mg/kg, PO, every 24 hours for 12 days (50 times the daily dose approved for use in dogs), 2 iguanas were given a single dose of meloxicam (0.2 mg/kg) PO, and 2 iguanas were given a single dose of meloxicam (0.2 mg/kg) IV. For iguanas receiving multiple doses, meloxicam was administered between 8 and 9 am each morning. Twenty-four hours after the last dose was administered, 1.5 mL of blood was collected from the caudal vein of each animal. A portion of each blood sample was placed in a tube containing EDTA and submitted for hematologic testing, and the remainder of each blood sample was placed in tubes containing heparin and submitted for biochemical testing and measurement of plasma meloxicam concentration. Hematologic testing included determination of PCV, total WBC count, differential leukocyte counts, and thrombocyte count. Total WBC count was determined with a 1:32 dilution of phloxine dye diluent,^g and differential leukocyte counts were performed manually on blood smears stained with Wright-Giemsa stain, as described.^14,15 Biochemical testing consisted of determination of sodium, potassium, chloride, and bicarbonate concentrations and anion gap in heparinized whole blood samples; all measurements were made with a portable analyzer.^h Plasma was obtained by means of centrifugation within 1 hour after blood sample collection, and plasma albumin, total protein, total calcium, phosphorus, and uric acid concentrations and creatinine kinase and aspartate aminotransferase activities were measured with a commercial analyzer.ⁱ Globulin concentration was calculated from measured total protein and albumin concentrations.

Necropsy and histologic examination—All 21 iguanas were euthanatized by means of IV administration of pentobarbitone^j 24 hours after the last meloxicam dose. Gross necropsy examinations were performed, and samples of the liver, both kidneys, the stomach, and any grossly abnormal tissues were placed in neutral-buffered 10% formalin and submitted for histologic examination. Samples were processed routinely and embedded in paraffin; 5-μm-thick sections were stained with H&E and examined microscopically.

Determination of plasma meloxicam concentration—Plasma meloxicam concentrations were measured by means of high-performance liquid chromatography performed with a commercial pump, sampler, and UV detector set at a wavelength of 355 nm.^k A 150 × 3-mm-internal-diameter, reverse-phase C18 column^l with a 2 cm X 4.0-mm guard column was used. The injection volume was 50 μL. The mobile phase consisted of a water—acetic acid (99:1 [vol/vol]; 65%) and acetonitrile (35%) mixture. Final solution pH was 3.2. An isocratic elution was performed. The flow rate was 0.7 mL/min. Plasma samples were extracted by pipetting 100 μL of plasma into a 1.5-mL microcentrifuge tube and adding 10 μL of piroxicam (1 mg/mL) in methanol, 30 μL of 1M hydrochloric acid, and 1.0 mL of diethyl ether. Samples were vortexed for 5 seconds and then centrifuged at 2,400 X g for 5 minutes at room temperature. The organic layer was transferred to borosilicate tubes. The remaining aqueous layer was resuspended in 1.0 mL of diethyl ether, vortexed for 10 seconds, and centrifuged. The second organic layer was added to the first extraction, and the combined solution was evaporated under nitrogen for at least 1 hour. Dried samples were stored at —4—C until analyzed by means of high-performance liquid chromatography. For analysis, dried samples were dissolved in 200 μL of the mobile phase and vortexed for 5 seconds, and 50 μL was injected in duplicate for analysis.

Selectivity of the method was confirmed by the lack of interfering peaks from endogenous compounds in blank plasma samples with the same retention times as meloxicam and piroxicam (internal standard). A calibration curve was prepared by adding meloxicam to the mobile phase and then fortifying banked iguana plasma to create known concentrations of meloxicam ranging from 0.01 to 25 μg/mL. For all calibration curves, the correlation coefficient for the best fit equation was ≥ 0.98. The limit of detection was 0.01 μg/mL, and the LOQ was 0.025 μg/mL. Extraction recoveries were 64.07% and 63.7%, respectively, following PO and IV administration. Five blood samples collected from an iguana not otherwise involved in the study were spiked with meloxicam (10 μg/mL), centrifuged, and stored for 2 hours, and meloxicam concentration was measured to identify any interference from lithium heparin in the plasma separator tubes.

Pharmacokinetic analysis—Plasma meloxicam concentrations after IV and PO administration were analyzed by means of noncompartmental analysis; standard software^m was used. The AUC from time 0 to the last measured concentration (AUC_0–cn) was calculated by means of the log-linear trapezoidal method; for this calculation, the last measured concentration was defined as the last concentration greater than the LOQ. The AUC from time 0 to infinity (AUC_0-∞) was calculated by adding the area under the terminal portion of the curve to the AUC_0–cn; the area under the terminal portion of the curve was estimated as Cn/γ_Z, where γ_Z represented the slope of the terminal portion of the curve and Cn represented the last measured concentration. Mean residence time, systemic Cl, t_1/2, γ_Z, and apparent VDs (VD_area and VD_SS) were calculated in accordance with statistical moment theory, as described.¹⁷ Maximum plasma concentration and time to maximum plasma concentration were also determined. The F following PO administration was calculated as the ratio of AUC_0-∞ following PO administration to the AUC_0-∞ following IV administration; values of F were converted to percentages. One iguana had plasma meloxicam concentrations following IV administration approximately 30 times greater than concentrations measured at comparable times in the other iguanas; data following IV administration in this iguana were not included in calculations.

Statistical analysis—The Shapiro-Wilk test was used to check whether data were normally distributed. A 2-tailed, paired Student t test was used to compare body weights measured before the study and just prior to euthanasia. Two-tailed, nonpaired Student t tests for samples with unequal variance were used to compare pharmacokinetic parameters obtained following IV versus PO administration, and between sexes. Standard software was used for all calculations.ⁿ Values of P < 0.05 were considered significant.

Results

Overall mean ± SE body weight was 469 ± 51 g, and body weight did not change significantly between the start and end of the study. Data for pharmacokinetic parameters calculated following PO and IV administration of single doses of meloxicam were normally distributed. There were no significant differences between PO and IV administration with regard to t_1/2, γ_z, AUC_0-cn, AUC_0-∞, the percentage of AUC_0-∞ that was extrapolated, VD_area, or Cl (Figure 1; Table 1). Mean residence time was significantly longer following PO administration than following IV administration. The F following PO administration was high (108 ± 49%). Results of analysis of control plasma samples indicated that there was no interference from lithium heparin in the plasma separator tubes. There were no significant differences associated with sex.

Mean ± SE plasma meloxicam concentrations in healthy green iguanas following PO (circles; n = 13) and IV (squares; 12) administration of a single dose (0.2 mg/kg). The LOQ of the meloxicam assay was 0.025 μg/mL, and the limit of detection was 0.01 μg/mL.

Citation: American Journal of Veterinary Research 71, 11; 10.2460/ajvr.71.11.1277

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Mean ± SE plasma meloxicam concentrations in healthy green iguanas following PO (circles; n = 13) and IV (squares; 12) administration of a single dose (0.2 mg/kg). The LOQ of the meloxicam assay was 0.025 μg/mL, and the limit of detection was 0.01 μg/mL.

Citation: American Journal of Veterinary Research 71, 11; 10.2460/ajvr.71.11.1277

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Mean ± SE plasma meloxicam concentrations in healthy green iguanas following PO (circles; n = 13) and IV (squares; 12) administration of a single dose (0.2 mg/kg). The LOQ of the meloxicam assay was 0.025 μg/mL, and the limit of detection was 0.01 μg/mL.

Citation: American Journal of Veterinary Research 71, 11; 10.2460/ajvr.71.11.1277

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Results of hematologic and biochemistry tests performed on 4 iguanas that received a single dose of meloxicam (0.2 mg/kg, PO or IV) were unremarkable (Table 2), compared with published reference ranges for healthy iguanas.^18–21 The 4 iguanas that received multiple high doses of meloxicam (1 or 5 mg/kg, PO, q 24 h for 12 days) had high total protein concentrations and high total WBC, heterophil, monocyte, and lymphocyte counts. Iguanas that received the higher dosage also had high uric acid concentrations.

For all 21 iguanas, necropsy results and results of histologic examination of the stomach, liver, and kidneys were unremarkable. One iguana had mild hepatic lipidosis but also had a good body condition with large fat bodies. Two other iguanas each had a single small bacterial granuloma in the kidney or pancreas that was considered clinically unimportant.

Discussion

Results of the present study suggested that administration of meloxicam at a dose of 0.2 mg/kg IV or PO in healthy green iguanas would result in plasma concentrations > 0.1 μg/mL for approximately 24 hours. Because pharmacodynamic and efficacy studies of meloxicam in iguanas have not been performed, the minimum plasma concentration necessary for analgesic effects is not known. Plasma meloxicam concentrations of 0.57 to 0.93 μg/mL in humans,²² 0.130 to 0.195 μg/mL in horses,²³ and 0.82 μg/mL in dogs²⁴ have been shown to induce antiinflammatory effects. Therefore, it is unclear whether a meloxicam dose of 0.2 mg/kg would induce analgesic effects in iguanas. Importantly, the pharmacokinetics of meloxicam following multiple-dose administration in iguanas was not determined in the present study, and long-term administration cannot be recommended until multiple-dose pharmacokinetic studies and additional safety studies are undertaken. Pharmacokinetics following IM injection was not determined in the present study; however, IM administration could be expected to result in plasma meloxicam concentrations similar to those seen after IV administration.

The dose of meloxicam used in the present study (0.2 mg/kg) was chosen on the basis of pharmacokinetic data for dogs²⁵ and 1 author's (SJD) clinical experience between 1994 and 2001. Our results suggested that PO or IV administration of a single dose of meloxicam (0.2 mg/kg) was well tolerated and did not cause obvious adverse effects. There was no evidence of vomiting, changes in food intake, or changes in body weight in any of the iguanas, and histologic evaluation of the stomach, liver, and kidneys did not reveal any abnormalities associated with NSAID administration.

It would have been preferable to randomize the routes of administration in the pharmacokinetics phase of the present study; however, the differing venipuncture schedules following IV versus PO administration coupled with limited technical manpower made this impractical. The lack of randomization means that we cannot rule out the possibility that carryover effects altered results obtained following IV administration. To our knowledge, however, autoinduction and autoinhibition have not been reported following administration of meloxicam in any species.

Because of the blood sampling schedule used in the present study, the elimination rate was calculated only on the basis of the last 2 time points, which is not ideal and results in a greater degree of uncertainty for calculated pharmacokinetic parameters. In addition, data for 1 iguana were not used in calculation of pharmacokinetic parameters following IV administration because measured concentrations were so high, compared with concentrations for the other iguanas. We believe that partial perivascular injection of meloxicam may have resulted in high local meloxicam concentrations in the tail that affected blood samples subsequently collected from the caudal vein. In general, the authors were confident that the technique used for IV administration via the caudal vein tail was effective for drug delivery and would be useful clinically. Nevertheless, the potential for extravascular injection and reduced accuracy in dosing when delivering small volumes to reptiles must be considered. In addition, blood from the caudal vein can pass through the kidneys (renoportal circulation) and the liver (hepatoportal circulation) prior to entering the systemic circulation, and the potential effects of this on our data were not determined. It would have been preferable to insert a jugular catheter for blood sample collection, but this would have required general anesthesia and a surgical procedure. Given the additional research procedures that these animals were to be subjected to after the pharmacokinetic study, jugular catheterization was not performed because of welfare concerns.

Six iguanas in the present study had a strong secondary peak in plasma meloxicam concentration between 12 and 23 hours after IV administration. A similar peak was suspected following PO administration, although it was not as evident because blood samples were collected less frequently, suggesting that this secondary drug peak was not route specific. Similar phenomena have been observed in dogs and rats in association with biliary recycling of drugs conjugated with glucuronide and reabsorbed.^26–28 Following IV administration of carprofen in dogs, the S-enantiomer metabolite undergoes enterohepatic recycling, whereas the R-enantiomer does not.²⁷ In dogs given tolfenamic acid PO, the parent drug is well absorbed from the duodenum and ileum, but the conjugated form is only absorbed following bacterial hydrolysis from the ileum.²⁶ Given the delays associated with intestinal transit and hydrolysis, the authors concluded that a long delay must exist between bile excretion of a conjugate and reabsorption of its free moiety from the ileum. Extrapolation to iguanas with their longer gastric and small intestinal emptying times²⁹ suggests that delayed enterohepatic recirculation of meloxicam could have been the cause of the secondary drug peak seen in the present study. Green iguanas, like other reptiles that possess a urinary bladder, are known to be able to modify the composition of urine across the bladder wall.³⁰ Thus, it may be possible that soluble drugs and their metabolites may be reabsorbed into the circulation along a concentration gradient across the bladder wall, leading to a secondary drug peak many hours after initial administration. Although not previously reported in lizards, this secondary drug peak phenomenon has been observed in chelonians. Greek tortoises (Testudo graeca) and Hermann's tortoises (Testudo hermanni) demonstrated secondary drug peaks 24 hours after IM injection of carbenicillin.³¹ A pharmacokinetic study³² of fluconazole in loggerhead sea turtles (Caretta caretta) also demonstrated a secondary plasma peak 48 hours after IM injection.

To our knowledge, there are no previous published studies of the pharmacokinetics of meloxicam in reptiles. However, the pharmacokinetics of meloxicam following IV administration has been studied in a variety of mammals and birds, including pigeons, ducks, turkeys, ostriches, chickens, mice, rats, guinea pigs, dogs, and humans.^22,25,33 Terminal half-lives following IV and PO administration in the present study were greater than those reported for any bird or mammal previously studied, other than male mice. Meloxicam Cl for iguanas in the present study was lower than values for most avian species (except chickens) and greater than values for rats, dogs, and humans. In addition, values for VDss in the present study were high, exceeded only by values reported for ostriches, male mice, and guinea pigs. Absorption following PO administration of meloxicam to green iguanas was essentially 100%.

The cause of the clinicopathologic changes noted following daily administration of high doses of meloxicam (1 or 5 mg/kg, PO) was unclear. Because of constraints regarding the number of animals included in the study and the number of procedures that could be performed on each animal, we were limited in our ability to perform toxicity studies at therapeutic drug doses or to assess changes in control animals. The lack of histologic abnormalities in the stomachs, livers, and kidneys of iguanas in the present study despite very high plasma drug concentrations suggested that drug-induced damage was unlikely; however, functional evaluations were not performed. Plasma uric acid concentrations were high in the 4 iguanas given meloxicam at the higher dosages but only exceeded the range previously reported for iguanas with normal renal function at the 5 mg/kg dose.³⁴ The lymphocytosis that was seen may have been associated with an infectious or inflammatory condition, but there was no evidence of this at necropsy. Nevertheless, until the clinical importance of these changes is known, we do not recommend administering meloxicam at a dose higher than 0.2 mg/kg in iguanas.

Temperature appears to cause variable effects on reptile pharmacokinetics. Amikacin pharmacokinetics is affected by temperature in gopher snakes (Pituophis melanoleucus catenifer) but not in ball pythons (P regius).^35,36 In gopher tortoises (Gopherus polyphemus), amikacin Cl is affected by temperature but half-life is not.³⁷ The iguanas in the present study were provided with a species-specific temperature range to facilitate thermoregulation, rather than a fixed ambient temperature. Although it is possible that variable environmental temperature may have affected metabolism and Cl of meloxicam in the present study, organ perfusion and function tend to remain near constant when lizards are provided with the ability to select a preferred body temperature from within their preferred temperature zone.^38,39