J. vet. Pharmacol. Therap. doi: /jvp SHORT COMMUNICATION H. K. KNYCH*, S. D. STANLEY*, R. M. ARTHUR & D. S. MCKEMIE*

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1 J. vet. Pharmacol. Therap. doi: /jvp SHORT COMMUNICATION Disposition of the anti-ulcer medications ranitidine, cimetidine, and omeprazole following administration of multiple doses to exercised Thoroughbred horses H. K. KNYCH*, S. D. STANLEY*, R. M. ARTHUR & D. S. MCKEMIE* *K.L. Maddy Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, CA, USA; Department of Veterinary Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA, USA; School of Veterinary Medicine, University of California, Davis, CA, USA Knych, H. K., Stanley, S. D., Arthur, R. M., McKemie, D. S. Disposition of the anti-ulcer medications ranitidine, cimetidine, and omeprazole following administration of multiple doses to exercised Thoroughbred horses. J. vet. Pharmacol. Therap. doi: /jvp The use of anti-ulcer medications, such as cimetidine, ranitidine, and omeprazole, is common in performance horses. The use of these drugs is regulated in performance horses, and as such a withdrawal time is necessary prior to competition to avoid a medication violation. To the authors knowledge, there are no reports in the literature describing repeated oral administrations of these drugs in the horse to determine a regulatory threshold and related withdrawal time recommendations. Therefore, the objective of the current study was to describe the disposition and elimination pharmacokinetics of these anti-ulcer medications following oral administration to provide data upon which appropriate regulatory recommendations can be established. Nine exercised Thoroughbred horses were administered 20 mg/kg BID of cimetidine or 8 mg/kg BID of ranitidine, both for seven doses or 2.28 g of omeprazole SID for four doses. Blood samples were collected, serum drug concentrations were determined, and elimination pharmacokinetic parameters were calculated. The serum elimination half-life was , and h for cimetidine, ranitidine, and omeprazole, respectively. Serum cimetidine and ranitidine concentrations were above the LOQ and omeprazole and omeprazole sulfide below the LOQ in all horses studied upon termination of sample collection. (Paper received 10 January 2016; accepted for publication 27 April 2016) Heather Knych, K.L. Maddy Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616, USA. hkknych@ucdavis.edu Anti-ulcer medications, such as cimetidine, ranitidine, and omeprazole, are commonly used in performance horses to treat gastric and duodenal ulcers. All three drugs decrease gastric acid production, cimetidine and ranitidine by blocking H 2 receptors and omeprazole by inhibiting proton pumps. They are all classified, by the Association of Racing Commissioners International (ARCI), as class 5 foreign substances, and as such their use is regulated in horse racing. While the pharmacokinetics of cimetidine, ranitidine, and omeprazole following a single administration have been reported (Sams et al., 1997; Smyth et al., 1990; Holland et al., 1997; Duran & Ravis, 1993), to the authors knowledge, there are no reports describing serum concentrations or the disposition of these drugs following multiple administrations, which is commonplace in performance horse practice. The objective of the current study was to describe serum concentrations and the disposition of cimetidine, ranitidine, and omeprazole following multiple oral administrations to horses, utilizing a highly sensitive liquid chromatography mass spectrometry (LC MS/MS) method currently employed by drugtesting laboratories to analyze biological samples collected from racehorses. Omeprazole sulfide concentrations were also determined as some jurisdictions use the metabolite as the target analyte to regulate the use of omeprazole. The common use of multiple administrations of these three drugs in performance horses warrants further study of these drugs to establish appropriate regulatory recommendations. Nine healthy exercised adult Thoroughbred horses including seven geldings and two mares (4 9 years of age; weight of kg) were used for each administration. Horses are exercised on a continual basis, and exercise continued throughout the study. The exercise regimen consists of 6 days a week of exercise, including 3 days on an Equigym â treadmill (Equigym, LLC, Lexington, KY, USA) and 3 days on a 1

2 2 H. K. Knych et al. mechanical walker (Equigym, LLC). Before beginning the study, horses were weighed and determined healthy and free of disease by physical examination, complete blood count, and a serum biochemistry panel. Horses did not receive any other medications for at least 2 weeks prior to commencement of this study. This study was approved by the Institutional Animal Care and Use Committee of Kentucky Equine Research (KER). Prior to drug administration, a 14-gauge catheter was aseptically placed in one external jugular vein for sample collection. A balanced crossover design was used with a 2-week washout in between treatments. Cimetidine tablets (Mylan Pharmaceuticals Inc, Morgantown, WV, USA) and ranitidine tablets (Amneal Pharmaceuticals of NY, Hauppauge, NY, USA) were administered suspended in water (60 ml) via a dosing syringe at doses of 20 mg/kg and 8.0 mg/kg BID for a total of seven doses for cimetidine and ranitidine, respectively. Omeprazole (Gastrogard Paste, Merial, Duluth, GA, USA) was administered at a dose of 2.28 g per horse ( mg/kg) SID for a total of four doses. Dose selection for all drugs was determined based on an informal survey of equine practitioners conducted by the Racing Medication and Testing Consortium. Horses were fed 1 h after dosing. Blood samples were collected at time 0, 1, 2, 4, 6, and 12 h after the first dose for cimetidine and ranitidine and at time 0, 1, 2, 4, 6, 12, and 24 h after the first dose for omeprazole. Following the last dose, samples were collected at 1, 2, 4, 6, 12, 24, 36, and 48 h for all three drugs. Additional samples were collected at 12 h intervals during the dosing period (immediately prior to drug administration). Prior to drawing each sample of blood, 10 ml of blood was aspirated from the catheter and T-port extension set and discarded. The catheter was flushed with 10 ml of a dilute heparinized saline solution (10 IU/mL) following each sampling. Blood samples (15 ml) were collected into serum separator tubes and placed on ice prior to centrifugation at g for 10 min. Serum was transferred into storage cryovials and stored at 20 C until shipped to the laboratory for analysis. Samples were shipped on dry ice and immediately transferred to a 20 C freezer upon receipt. Omeprazole reference standard was obtained from Cerilliant Corporation (Round Rock, TX, USA), omeprazole sulfide from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and cimetidine and ranitidine from Sigma-Aldrich (St. Louis, MO, USA). Deuterated standards of the same molecule were used for all analyses (Toronto Research Chemicals, Toronto, ON, Canada). Omeprazole and omeprazole sulfide were extracted from serum using a liquid/liquid extraction procedure with a mixture of methyl tert-butyl ether:methylene chloride (Wang et al., 2005). Cimetidine and ranitidine were extracted from serum with solid-phase extraction using a mixed mode SPE column of reverse phase and cation exchange (Zendelovska & Stafilov, 2003). All three used 0.5 ml volumes of serum. Serum calibrators and quality control samples were prepared by dilution of working standard solutions with drug-free equine serum. Quantitative analysis of serum was performed on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA) having 1100 series liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA). Product masses and collision energies of each analyte were optimized by infusing the standards into the TSQ Vantage. Chromatography employed an ACE 3 C18 10 cm mm column (Mac-Mod Analytical, Chadds Ford, PA, USA) and a linear gradient of acetonitrile in water with a constant 0.2% formic acid, at a flow rate of 0.40 ml/min. Detection and quantitation were conducted using selective reaction monitoring. The response for the product ion(s) for all analytes and internal standards were plotted and peaks at the proper retention time integrated using Quanbrowser software (Thermo Scientific, San Jose, CA, USA). The drugs were quantitated by linear regression analysis. A weighting factor of 1/X was used for all calibration curves. The response for all drugs was linear and gave correlation coefficients (R 2 ) of 0.99 or better. Accuracy was reported as percent nominal concentration, and precision was reported as percent relative standard deviation. The technique was optimized to provide a limit of quantitation (LOQ) of 0.1 ng/ml (omeprazole and omeprazole sulfide), and 0.05 ng/ml (ranitidine and cimetidine) and a limit of detection (LOD) of Table 1. Accuracy and precision values for LC MS/MS analysis of cimetidine, ranitidine, omeprazole, and omeprazole sulfide in equine serum Drug Concentration (ng/ml) Intraday accuracy (% nominal conc) Intraday precision % relative SD) Interday accuracy (% nominal conc) Interday precision (% relative SD) Cimetidine Ranitidine Omeprazole Omeprazole sulfide

3 Disposition of anti-ulcer medications in horses 3 approximately 0.03 ng/ml (omeprazole and omeprazole sulfide), 0.01 ng/ml for ranitidine and ng/ml for cimetidine. Concentration data were log-transformed to meet the assumptions of normality. Noncompartmental analysis was used for determination of pharmacokinetic parameters for all compounds using commercially available software (Phoenix WinNonlin version 6.3, Pharsight, Cary, NC, USA). The area under the curve was calculated using the log-linear trapezoidal rule and extrapolation to infinity using the last measured plasma concentration divided by the terminal slope k z. Accuracy and precision for the assays were considered acceptable based on Food and Drug Administration guidelines for bioanalytical method validation (Table 1). The semilog plots of serum cimetidine, ranitidine, omeprazole, and omeprazole sulfide concentrations over time are depicted in Fig. 1a c. serum concentrations for all drugs are listed in Table 2, and pharmacokinetic parameters following drug administration are listed in Tables 3 and 4. Concentrations varied widely between horses at each time point for all drugs. The AUC did not increase substantially between the first and last dose for any of the drugs as indicated by a median accumulation index of 1.47, 1.50, and 1.02 for cimetidine, ranitidine, and omeprazole, respectively. The serum elimination half-life was , and for cimetidine, ranitidine, and omeprazole, respectively. Serum cimetidine and ranitidine concentrations were still above the LOQ (0.05 ng/ml) of the assay at 72-h postadministration of the last dose in all horses studied. Omeprazole and omeprazole sulfide concentrations were below the LOQ in all horses upon termination of sample collection. Long-term administration of anti-ulcer medications in racehorses is commonplace. As the use of this drug is regulated in racehorses and to the best of the authors knowledge, there are no reports describing the pharmacokinetics of cimetidine, ranitidine, and omeprazole following multiple oral administrations, the primary objective of the current study was to describe the disposition of these three drugs in horses to provide data upon which appropriate regulatory recommendations can be made. As the goal of this study was to generate data for establishment of regulatory recommendations, we chose to focus primarily on elimination pharmacokinetics. There was no effect of treatment order on pharmacokinetic parameters. The terminal serum half-life of cimetidine and ranitidine in the current study was prolonged ( h and ) compared to previous reports describing administration to horses (cimetidine: h (3.3 and 10 mg/kg PO), Smyth et al., 1990; 92.4 min (4.0 mg/kg intragastric), Sams et al., 1997; ranitidine: 84.7 min (2.2 mg/kg PO), Holland et al., 1997). This difference is likely attributable to the more sensitive analytical assay utilized in the current study (LOQ of 0.05 ng/ml) as compared to the previous studies (250 ng/ml, Sams et al., 1997; 30 ng/ml, Holland et al., 1997) or in the case of cimetidine, differences in sampling protocols. Blood samples were collected for a prolonged period of time, in the current study (48-h postadministration of the final dose) relative to the study by Sams et al. (1997) (8-h postdose). Being able to quantitate concentrations for a longer period of time requires less extrapolation of the terminal elimination portion of the plasma concentration time curve, likely allowing for a more accurate calculation of the elimination half-life. The terminal elimination half-life for omeprazole ranged from 3.02 to 6.33 h, which Figure 1. Average SD serum concentrations of (a) cimetidine, (b) ranitidine and (c) omeprazole and omeprazole sulfide, concentrations following oral administration of 20 mg/kg BID (97 doses) of cimetidine or 8 mg/kg BID (seven doses) or 2.28 g SID (four doses) of omeprazole to nine exercised Throughbred horses.

4 4 H. K. Knych et al. Table 2. Mean SD serum cimetidine, ranitidine, and omeprazole concentrations at various time postoral administration to nine exercised Thoroughbred horses. Times are postadministration of the first dose Time (h) Cimetidine 20 mg/kg Ranitidine 8 mg/kg Omeprazole 2.28 g SID (4 doses) Omeprazole Sulfide 2.28 g SID (4 doses) 0 ND ND ND ND ND ND Table 3. Pharmacokinetic parameters following multiple oral administration of cimetidine and ranitidine to nine exercised Thoroughbred horses. All values in this table were generated using noncompartmental analysis Parameter Cimetidine 20 mg/kg Ranitidine 8 mg/kg Lambda z (1/h) Serum t 1/2k (h)* AUC tau (hng/ml) C avg (ng/ml) C last (ng/ml) Accumulation Index *Harmonic mean; Lambda z, terminal slope; t 1/2k, half-life k z ; AUC tau, area under the plasma concentration time curve during a dosing interval; C avg, average serum concentration; C last, last measured serum concentration; Accumulation index was determined by use of the equation (AUC[12 or 24], s)/(auc[1, s]), where dose 7 or 4 and 1 are the last and first dose, respectively. is in agreement with previous reports ( h, Product Information, Merial Limited). In the current study, we have presented the AUC tau and accumulation index; however, it is important to note that these parameters should be interpreted with caution due to the sparse sample collection in the initial period postdrug administration. The presently reported study provides data that can be utilized to establish appropriate regulatory recommendations. Based on the increased sensitivity of currently used analytical instrumentation in drug-testing laboratories, prolonged detection times are possible for cimetidine and ranitidine necessitating quantitative regulatory thresholds. Additionally, if Table 4. Pharmacokinetic parameters for omeprazole and omeprazole sulfide following administration of 2.28 g BID for four doses of omeprazole paste to nine exercised Thoroughbred horses. All values in this table were generated using noncompartmental analysis Parameter prolonged administration protocols are used, an extended withdrawal time may be necessary prior to competition. ACKNOWLEDGMENTS Omeprazole Omeprazole Sulfide Lambda z (1/h) Serum t 1/2k (hr)* AUC tau (hng/ml) C avg (ng/ml) C last (ng/ml) Accumulation Index *Harmonic mean; Lambda z, terminal slope; t 1/2k, half-life k z ; AUC tau, area under the plasma concentration time curve during a dosing interval; C avg, average serum concentration; C last, last measured serum concentration; Accumulation index was determined by use of the equation (AUC[12 or 24], s)/(auc[1, s]), where dose 7 or 4 and 1 are the last and first dose, respectively. Funding for the drug administration portion of this study was provided by the Kentucky Equine Drug Research Council, and funding for determination of drug concentrations was provided by the Racing Medication and Testing Consortium. The authors would like to thank Sandy Yim, Sheena Mouton, Dr. Byran Waldridge, and the Kentucky Equine Research staff for technical assistance.

5 Disposition of anti-ulcer medications in horses 5 REFERENCES Duran, S.H. & Ravis, W.R. (1993) Comparative pharmacokinetics of H2 antagonists in horses. Proceedings of the 11th Annual Veterinary Medical Forum, American College of Veterinary Internal Medicine, pp Holland, P.S., Ruoff, W.W., Brumbaugh, G.W. & Brown, S.A. (1997) Plasma pharmacokinetics of ranitidine HCL in adult horses. Journal of Veterinary Pharmacology and Therapeutics, 20, Sams, R.A., Gerken, D.F., Dyke, T.M., Reed, S.M. & Ashcraft, S.M. (1997) Pharmacokinetics of intravenous and intragastric cimetidine in horses I. Effects of intravenous cimetidine on pharmacokinetics of intravenous phenylbutazone. Journal of Veterinary Pharmacology and Therapeutics, 20, Smyth, G.B., Duran, S., Ravis, W. & Clark, C.R. (1990) Pharmacokinetic studies of cimetidine hydrochloride in adult horses. Equine Veterinary Journal, 22, Wang, J., Wang, Y., Fawcett, J.P., Wang, Y. & Gu, J. (2005) Determination of omeprazole in human plasma by liquid chromatographyelectrospray quadrupole linear ion trap mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 39, Zendelovska, D. & Stafilov, T. (2003) Development of an HPLC method for the determination of ranitidine and cimetidine in human plasma following SPE. Journal of Pharmaceutical and Biomedical Analysis, 33,