Meloxicam [4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1, 2-benzothiazine-3-carboxamide-1,1-dioxide] is a potent non-steroidal anti-inflammatory drug (NSAID) of the oxicam group.These structurally related drugspossess analgesic, antipyretic and anti-inflammatory properties, with low ulcerogenic potency and less local tissue irritation in comparison to other NSAIDs such as diclofenac, indomethacin and piroxicam[1]. Meloxicam is a drug of choice in the management of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis by relieving pain and reducing inflammation in humans[2]. Meloxicam inhibits prostaglandin synthesis via selective inhibition of cyclooxygenase-2 enzyme and thus shows superior gastrointestinal tolerability than other NSAIDs[3]. The drug is available as either injectable or oral formulations in many countries worldwide[4].

  Meloxicam has considerably varied pharmacokinetic parameters among species. For example, the elimination half-life ranged from 20 - 24 hours in humans[5] to about 8.54 hours in horses[6]. Recently, many positive findings were reported for meloxicam in racing camels in U.A.E. Veterinary records showed that the animals received a therapeutic dose of meloxicam 20 days before the race events. A previous study has highlighted a detection period of 10 days for meloxicam in camel plasma[7]. It has been noted that no more blood samples were collected after 10 days and the reported analytical method lacks the sensitivity required to detect meloxicam at lower trace levels.

  Therefore, the objective of the present study was to characterize the plasma pharmacokinetics, urinary excretion profile and metabolism after administration of a single IV dose of meloxicam to camels. Another goal of the study was to advise the field veterinarians about the detection and withdrawal time period for meloxicam and its metabolites in camels used in racing employing the latest high resolution bench-top orbitrap mass spectrometry (LC-MS/MS) with an in-house developed solid phase extraction method.

Materials and Methods

Animals

  Four healthy male camels (Camelus dromedaries) aged 5 – 10 years and weighing approximately 500 kg were used in this study. The camels were tagged, dewormed and housed in shaded pens. They were fed with hay and lucerne, with water allowed ad libitum. The health status of these experimental animals was regularly monitored throughout the experiment by project veterinarians. The study had the relevant ethical clearance by the local animal ethics committee to undertake the work.

Experimental chemicals

  Meloxicam at 20 mg/mL IV solution (Metacam, Boehringer Ingelheim Vetmedica) was purchased as the administration drug under investigation. All organic solvents were of HPLC grade and purchased from Fisher Scientific. Meloxicam as pure reference standard, d₃-flunixin and β-glucuronidase from Helix pomatia (type HP2) were purchased from Sigma Chemical Co.Ltd.

Treatment and sample collection

  Meloxicam was administered as an intravenous single dose of 0.5 mg/kg (body weight) into the right jugular vein. Venous blood samples were collected from the jugular vein at 0 min (pre-injection), 5, 10, 15, 30, and 45 min and 1, 2, 4, 6, 8, 12, 24 hours post medication through an in-dwelling cannula on the opposite side. Blood samples were collected once daily for the next 30 days. The samples were centrifuged at 4000 ɡ at room temperature for 10 min and plasma was separated and stored at -20 °C until analysis. In addition, urine was collected as morning and evening samples as the animal disposes during the same period.

Sample preparation

  Plasma and urine samples were analyzed by use of liquid chromatography-orbitrap mass spectrometry. Solid phase extraction (SPE) was carried out using automated extraction modules (RapidTrace, Biotage) using mixed-mode extraction cartridges that combine both non-polar (C₈) and strong anion exchange retention (-NR₃+) mechanisms (Isolute HAX, Biotage). A volume of 2 mL of plasma was combined with 20 μl internal standard (flunixin-d3 [1 μg/mL]). The extraction cartridge was conditioned with methanol (2 mL) and 0.05 M ammonium acetate (2 mL). The mixed sample of plasma and internal standard was loaded on to the cartridge and allowed to pass through at a slow rate. The cartridge was then washed with a mixture of methanol and 0.05M ammonium acetate (1 mL [1:9]) and allowed to dry under nitrogen for 5 min. Meloxicam was eluted with 2% acetic acid in methanol (5 mL) into clean test tubes. The organic solvent was evaporated under nitrogen at 50 °C. Samples were reconstituted with the analytical method mobile phase and 10 μL is injected into the LC-MS/MS system.

  Similarly, a qualitative analysis to study the metabolites and excretion of meloxicam in urine was performed. Duplicate aliquots of 5 mL of camel urine were used for extraction. One aliquot was adjusted to pH 5 and enzyme hydrolyzedusing β-glucuronidase by incubating at 56 °C for 2 hours while the other aliquot was not subjected to enzymatic hydrolysis. To both aliquots, 50 μL internal standard (flunixin-d3 [1 μg/mL]) was added, pH was adjusted to 7.0, and centrifuged at 4000 ɡ at room temperature for 10 min. Thereafter, 4 mL of the clear supernatant sample from each aliquot was extracted separately with the described SPE procedure.

LC-MS/MS analysis

  Detection of meloxicam was performed by an in-house developed and validatedLC–MS / MS using high pressure liquid chromatography system (Ultimate 3000, Thermo Dionex) coupled with orbitrap high resolution mass analyser (Q Exactive, Thermo Scientific). The mobile phase consisted of acetonitrile and 1% formic acid at a flow rate of 0.6 mL/min, run in a multi-step gradient mode. Separation was achieved by using reversed phase C18 column 4.6 mm x 150 mm, 3.5 micron (Zorbax Eclipse, Agilent Technologies). The mass spectrometer was operated in positive ion mode and the total run time was 8.4 minutes.

  Calibration curves were prepared for the quantitative determination of meloxicam in plasma samples obtained after IV administration. The calibrators ranged from 1.0 to 2000 ng/mL. Two quality control sample spiked at 10 and 50 ng/ml of meloxicam in plasma were also prepared. Calibrators and quality controls were prepared independently from separate weightings of the drug standard. All calibrators, quality control sample and test samples were analyzed in duplicate by LC-MS/MS. The peak area ratios of meloxicam to the internal standard versus the concentration of the calibrators were fitted using linear regression. The response for the assay method was linear over the designed calibration range (r² = 0.998). The limit of quantitation was 1 ng/mL. The within-day and day to day precision were ˂ 10%.

Identification of metabolites

  Aliquots of urine samples taken before and after the IV administration were extracted by SPE and analyzed using LC-MS/MS system applying Full MS, All-Ion-Fragment (AIF) and Data-Independent-Acquisition (DIA) scan functions of the mass analyzer. Meloxicam metabolites were identified by comparison of the obtained chromatographic peaks and spectra against reported spectra with the aid of commercial available spectral data-base (Association of Official Racing Chemists).

Pharmacokinetic Analysis

  Pharmacokinetic parameters were determined for each animal individually from plasma drug concentration versus time profile using a computerized pharmacokinetic and drug disposition program (Kinetica Version 5.1 SP1, Thermo). The best fitting was achieved by two compartment open model based on the statistical moments theory[8,9]. All data were analysed using SPSS package. For each parameter, the mean and its standard deviation (SD) were obtained from the parameters estimates of four animals. The area under the plasma concentration-time curve (AUC) was calculated by the trapezoidal method up to the last measured concentration.

Results

  The pharmacokinetics (PK) of meloxicamafter a single dose IV administration (0.5 mg/kg) in camels was best fitted with two compartmental open model and followed the first order rate kinetics.The graphical representation of the mean ± SDplasma drug concentration (μg/mL) of meloxicam in camels (n = 4) versus time is given in (Figure.1). Pharmacokinetic parameters estimates of meloxicam (mean ± SD) are presented in Table 1.

Figure 1: Time course graph showing plasma concentrationprofile of meloxicam in camels following an intravenous dose of 0.5 mg/kg (body weight). Values are presented as mean ± standard deviation.

Table 1: Pharmacokinetic parameters of meloxicam in camels (n = 4) after a single intravenous administration at a dose of 0.5 mg/kg (body weight). Data are presented as mean ± SD.

  The plasma drug profile (Figure.1) indicates an initial distribution phase of meloxicam with a relatively long distribution half-life (t_1/2α) of 0.863 ± 0.131 h. The distribution phase was followed by a very slow elimination phase with a very long terminal elimination half-life (t_{1 / 2β}) of 46.952 ± 4.694 h. The mean plasma drug concentration levels of meloxicam were still above the detection limit (>100 pg/mL) after 25 days of administration. The limit of quantitation (LOQ) of melociam in plasma was found to be 1ng/mL. The estimate of the extrapolated initial drug concentration (C_o) was 1.572 ± 0.391 μg/mL. The body clearance (Cl_T) was 5.587 ± 1.655 mL/kg/h and area under curve (AUC_0-t) was 57.665 ± 14.788 μg.h/mL.

  The urine samples evidenced the presence of two metabolites: 5’-hydroxymethylmeloxicam (m/z 368.0369) and 5’-carboxymeloxicam (m/z 382.0162), separated and characterized by the proposed LC-MS/MS method in positive ion mode, at retention times (RT) 5.86 min and 6.26 min respectively, as shown in (Figure.2.A,B). Meloxicam could be detected up to 25 days after administration in plasma with the limit of detection of 100 pg/mL. The metabolites 5’-carboxymeloxicam and 5’-hydroxymethylmeloxicam could be detected upto 17 days and 27 days in urine samples respectively. No significant changes were observed in the concentration of meloxicam and its metabolites in urine after enzymatic hydrolysis, indicating that meloxicam and its metabolites are excreted unconjugated.

Figure 2: Original chart recordings of LC-MS/MS extracted ion chromatogram and mass spectra of (A) 5’-hydroxymethylmeloxicam (RT = 5.86 min) and (B)5’-carboxymeloxicam (RT = 6.28 min) obtained from extracted camel urine after intravenous administration of meloxicam.

Discussion

  Meloxicam is a commercially available anti-inflammatory drug widely used to suppress pain[10]. Meloxicam has been found to be well absorbed and cleared almost exclusively by metabolic pathways in mice, rats, mini-pigs, baboons and human[11,12]. Meloxicam pharmacokinetics in camels has been previously reported[7]; however, the present study employed more frequent and longer blood sampling up to 30 days after drug administration. The sensitive analytical method developed enabled quantitation of meloxicam at very low levels (1 ng/mL) and is about ten times lower than the reported LOQ of 10 ng/mL.

  The mean ± SD plasma level of meloxicam was 1.41 ± 0.20 μg/mL at 0.05 h after IV administration, which declined rapidly to 0.7 ± 0.33 μg/mL after 3 hours, and thereafter the drug concentration declined to a steady state level. The distribution half-life (t_1/2α) and the elimination half-life (t_1/2β) of meloxicam in this study were 0.863 ± 0.131 h and 46.952 ± 4.694 h respectively, and are found to be much longer in camels compared to other species. The reported half-life of meloxicam in sheep and goat were 10.85 ± 1.21 and 6.73 ± 0.58 h, respectively[13]. Relatively shorter elimination half-life has been reported in avian species[14] while a half-life of 20 h has been reported in human[5]. In a short period experiment with less sampling intervals at the elimination phase, a shorter half-life of 40.2 ± 16.8 h for meloxicam has been reported in camels[7].

  The volume of distribution (Vd) in the present study was 371.940 ± 86.658 mL/ kg.This is more than the Vd reported in goat 276 mL/kg[15] and cattle 171 mL/kg[16,17]. These findings indicate that the drug is well distributed in camels emphasizing a therapeutic treatment of camels with meloxicam by a daily single dose administration.

  The present results have demonstrated that the proposed analytical method was highly sensitive that it could detect meloxicam in plasma at low levels at about 100 pg/mL after 25 days of administration. The calculated total body clearance (Cl_T) was 5.587 ± 1.655 mL/kg/h and it is found to be lower than that reported for horses (34.7 mL/kg/h)[6], piglets (61 mL/kg/h)[18], dogs (10 mL/kg/h)[9] and goats (22.6 mL/kg/h)[12], indicating a slower elimination in camels in comparison to other species. This is also evidenced with a comparatively high AUC_0-t in this study (57.665 ± 14.788 μg.h/mL) which also implies that the drug is eliminated at a lower rate in camels in comparison to other reported species[15-17,19].

Figure 3: Original chart recordings of LC-MS/MS extracted ion chromatogram and mass spectra of meloxicam obtained from extracted camel urine after intravenous administration of meloxicam.

  The ESI-MS/MS product ion spectrum of meloxicam (m/z 352.0420) shows two diagnostic fragment ions (Figure. 3). The protonation on the benzothiazine nitrogen heteroatom and cleavage of the benzothiazine-3-carboxamide bond without any proton transfer leads to formation of distinct m/z 141.0117. Furthermore, protonation of the amide nitrogen atom, two proton transfer and cleavage of amide linkage leads to the formation of m/z 115.0328[20]. The pharmacokinetics of meloxicam has been well studied in horses and the drug and twoof its metabolites could be detected for 72 hours[21]. In a previously published study on camels, only one metabolite of meloxicam namely, 5’-hydroxymethyxlmeloxicam has been reported and detected up to 10 days in plasma[7]. The present study shows that 5’-hydroxymethylmeloxicam (m/z 368.0369) was detected in urine samples up to 27 days after administration. In addition, further oxidation to this metabolite was observed resulting in the formation of 5’-carboxymeloxicam (m/z 382.0162) that could be detected up to 17 days. The two metabolites have been previously detected in humans, rats, mice, mini-pigs[9], and thoroughbred horses[20,22].

Conclusion

  The results obtained in the current study revealed that meloxicam followed the first order rate kinetics and it could be best fitted to a two compartmental open model. Two main metabolites could be isolated and detected from camel urine samples following IV administration of meloxicam. The main objective of this study was to investigate the pharmacokinetics of meloxicam and more over to find a withdrawal time following therapeutic administration for the practicing veterinarians. The proposed LC-MS/MS method was highly sensitive, with a limit of detection of 100 pg/mL, which could easily detect meloxicam in camel plasma for 25 days after administration. As such, a very long elimination half-life of meloxicam was observed in the present study. Hence, it is advised to withhold the use of meloxicam in camels for at least 30 days prior to racing.

Conflict of interest statement

  Boehringer Ingelheim Middle East & North Africa (Scientific Office) supplied Metacam (20 mg/mL solution for injection) used in this study. Boehringer Ingelheim Middle East and North Africa (Scientific Office) played no role in the study design nor in the collection, analysis and interpretation of data, nor in the decision to submit the manuscript for publication. None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

1. 1. Engelhardt, G., Homma, D., Schlegel, K., et al. Anti-inflammatory, analgesic, antipyretic and related properties of meloxicam, a new non-steroidal anti-inflammatory agent with favorable gastrointestinal tolerance. (1995) Inflamm Res 44(10): 423-433.

   Pudmed || Crossref || Others

2. 2. Sweetman, S.C., Martindale: The Complete Drug Reference, Thirty Fourth Edn. (2005) Pharmaceutical Press, London, pp. 56.

   Pudmed || Crossref || Others

3. 3. Vane, J.R., Botting, R.M. New insights into the mode of action of anti-inflammatory drugs. (1995) Inflamm Res 44(1): 1-10.

   Pudmed || Crossref || Others

4. 4. Churchill, L., Graham, A.G., Shih, C.K., et al. Selective inhibition of human cyclo-oxygenase-2 by meloxicam. (1996) Inflammo Pharmacology 4(2): 125-135.

   Pudmed || Crossref || Others

5. 5. Davies, N.M., Skjodt, N.M. Clinical pharmacokinetics of meloxicam. A cyclo-oxygenase-2 preferential nonsteroidal anti-inflammatory drug. (1999) Clin Pharmacokinet 36(2): 115-126.

   Pudmed || Crossref || Others

6. 6. Toutain, P.L., Cester, C.C. Pharmacokinetic-pharmacodynamic relationships and dose response to meloxicam in horses with induced arthritis in the right carpal joint. (2004) Am J Vet Res 65(11): 1533-1541.

   Pudmed || Crossref || Others

7. 7. Wasfi, I.A., Al Ali, W.A., Agha, et al. The pharmacokinetics and metabolism of meloxicam in camels after intravenous administration. (2012) J Vet Pharmacol Ther 35(2): 155-162.

   Pudmed || Crossref || Others

8. 8. Gabaldi, M., Perrier, D., Pharmacokinetic, Second Edn. Marcel Dekker Inc., (1982)New York, pp. 1-494.

   Pudmed || Crossref || Others

9. 9. Martinez, M.N. Non compartmental methods of drug characterization: statistical moment theory. (1998) J Am Vet Med Assoc 213(7): 974-980.

   Pudmed || Crossref || Others

10. 10. Allen, K. A., Coetzee, J.F., Edwards-Callaway, et al. The effect of timing of oral meloxicam administration on physiological responses in calves after cautery dehorning with local anesthesia. (2013) J Dairy Sci 96(8): 5194-5205.

    Pudmed || Crossref || Others

11. 11. Busch, U., Schmid, J., Heinzel, G., et al. Pharmacokinetics of meloxicam in animals and the relevance to humans. (1998) Drug Metab.Dispos 26(6): 576-584.

    Pudmed || Crossref || Others

12. 12. Schmid, J., Busch, U., Heinzel, G., et al. Pharmacokinetics and metabolic pattern after intravenous infusion and oral administration to healthy subjects. (1995) Drug Metab Dispos 23(11): 1206-1213.

    Pudmed || Crossref || Others

13. 13. Shukla, M., Singh, G., Sindhura, B.G., et al. Comparative plasma pharmacokinetics of meloxicam in sheep and goats following intravenous administration. (2007) Comp Biochem Physiol C Toxicol Pharmacol 145(4): 528-532.

    Pudmed || Crossref || Others

14. 14. Baert, K., De Backer, P. Comparative pharmacokinetics of three non-steroidal anti-inflammatory drugs in five bird species.Comp. Biochem. Physiol. (2003) Comp Biochem Physiol C Toxicol Pharmacol 134(1): 25-33.

    Pudmed || Crossref || Others

15. 15. Wani, A.R., Roy, R.K., Ashraf, A., et al. Pharmacokinetic studies of meloxicam after its intravenous administration in local goat (Capra hircus) of Assam. (2013) Vet World 6, 516-520.

    Pudmed || Crossref || Others

16. 16. Coetzee, J.F., KuKanich, B., Mosher, R., et al. Pharmacokinetics of intravenous and oral meloxicam in ruminant calves. (2009) Vet Ther 10(4): 67-71.

    Pudmed || Crossref || Others

17. 17. Coetzee, J.F., Mosher, R.A., KuKanich, B., et al. Pharmacokinetics and effect of intravenous meloxicam in weaned Holstein calves following scoop dehorning without local anesthesia. (2012) BMC Vet Res 8:153.

    Pudmed || Crossref || Others

18. 18. Fosse, T.K., Haga, H.A., Hormazabal, V., et al. Pharmacokinetics and pharmacodynamics of meloxicam in piglets. (2008) J Vet Pharmacol Ther 31(3): 246-252.

    Pudmed || Crossref || Others

19. 19. Sinclair, M.D., Mealey, K.L., Matthews, N.S., et al. Comparative pharmacokinetics of meloxicam in clinically normal horses and donkeys. (2006) Am J Vet Res 67(6): 1082-1085.

    Pudmed || Crossref || Others

20. 20. Dumasia, M.C., Bruce, S., Schofield, E. Detection of COX-2 selective NSAIDs: Biotransformation and identification of in vivo metabolites of meloxicam in equine urine. (2002) Proceedings of the 14th International Conference of Racing Analysts and Veterinarians, Florida, USA, 2002, pp. 343-348.

    Pudmed || Crossref || Others

21. 21. Toutain, P.L., Reymond, N., Laroute, V., et al. Pharmacokinetics of meloxicam in plasma and urine of horses. (2004) Am J Vet Res 65(11): 1542-1547.

    Pudmed || Crossref || Others

22. 22. de Kock, S.S., Rodgers, J.P., Jonker, L.. LC/MS/MS study of the metabolism and excretion of meloxicam in the horse. (2002) Proceedings of the 14th International Conference of Racing Analysts and Veterinarians, Florida, USA, pp. 135-141.

    Pudmed || Crossref || Others