NOVEMBER 2022
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squamous and glandular portions of the stomach were videoed, and photographs of the greater curvature, lesser curvature and pylorus were taken to be evaluated for lesion grading. One internal medicine specialist performed lesion grading for the entirety of the study and reviewed gastroscopy, videos and photographs. It is noted the internist was not blinded to treatment day.
Pharmacokinetic analysis Six of 23 horses were enrolled in both the efficacy study and the pharmacokinetic study. For the pharmacokinetic data,
blood was drawn prior to dosing (Day 2) and at 6, 12, 24, 48, 72, 96, 120, 144 and 168 h following the first dose of omeprazole. Approximately 6 ml of blood was drawn per sample timepoint, and serum was harvested and frozen for pharmacokinetic analysis. Pharmacokinetic analysis included LC/MS/MS detection of
omeprazole at each timepoint. Omeprazole was measured in equine serum using a validated LC/MS/MS assay carried out in the Pharmacology Shared Resource Laboratory at the CSU Veterinary Teaching Hospital. The instrumentation included an Applied Biosystems 3200 Q-TRAP triple quadrupole mass spectrometer coupled to an Agilent 1200 HPLC system and HTC-PAL autosampler. Unknown and quality control (QC) samples were prepared by adding known amounts of omeprazole (0.5–250 ng/ml) into blank serum. Samples (unknowns, standards and QCs) were prepared for analysis by mixing 50 ll of serum sample with 5 ll bupivicaine (10 ng/ ml, internal standard) and adding 100 ll acetonitrile followed by vortex mixing for 5 min to precipitate proteins. Samples were then centrifuged for 15 min at 13,300 rcf and the resulting supernatant collected and transferred to and HPLC vial with a low volume insert. Sample injection volume was 20 ll (using a 10 ll loading loop), and chromatography was carried out using a Waters Sunfire C18 5 lm column (4.6 9 50 mm) and a solvent system consisting of acetonitrile with 0.1% formic acid (solvent A) and Milli-Q water with 0.1% formic acid (solvent B). Omeprazole and bupivicaine (internal standard) were eluted using a gradient starting at 5 solvent A:95 solvent B for the first 1.0 min and then transitioning linearly to 95 solvent A:5 solvent B over a 2-min period and holding until 4 min when the original 5 solvent A:95 solvent B was re-established over a 1-min period and held for the remainder of the 6-min total run time. The mass spectrometer was operated in positive ion mode with an ion spray voltage of 5500 V and a source temperature of 550°C. Multiple reaction monitoring (MRM) analysis was carried out for omeprazole by monitoring ion transitions of 346.1 m/z? 198.0 m/z and 346.1 m/z?168.0 m/z and for bupivicaine at 289.3 m/z?140.2 m/z. MRM conditions were optimised using internal algorithms, and both Q1 and Q2 were operated at unit resolution. Assay performance was monitored using QC samples at 3 concentrations (2.5, 10, and 75 ng/ml) and
showed an accuracy and precision (%CV) of 94.1% 4.1% with a lower limit of quantitation of 0.5 ng/ml. All pharmacokinetic (PK) analyses were done using
noncompartmental analysis [PhoenixWinNonlin: Certara USA Inc.]. Cmax was calculated per horse, and means and standard deviations (SD) were calculated for all horses.
Statistical analysis Statistical analysis was used to evaluate ESGD and EGGD scoring and to compare pre- and post-study CBC/SBP data
and bodyweights [SAS v9.4: SAS Institute Inc.]. The data on Day 0, Day 14 and Day 28 were considered as repeated measures data. If the data met the assumption of normality, the analysis was performed using a repeated measures ANOVA and described using means and standard deviations. If the data did not meet the normality assumption, it was described using medians and first and third quartiles and was analysed using Friedman’s test. Post hoc Dunn’s test was used to perform pairwise comparisons. To compare multiple blood parameters between pre- and post-test, PROC MULTTEST was used to adjust the P-values for Bonferroni correction. A P- value of 0.05 was used to determine statistical significance.
Results
Clinical efficacy By Day 14, five of 23 horses (22%) had healed ESGD lesions. Of those, three horses continued to have healed ESGD lesions by Day 28, whereas the remaining horses had Grade 1 ulcers on Day 28. By Day 28, a total of eight horses (35%) demonstrated healing of ESGD lesions. Seventy-eight percent (18 of 23) of horses showed improvement in their ESGD scoring by Day 14. Three horses showed no improvement in ESGD scoring, and two horses showed worsening of ESGD grading by Day 14 (from grade 2 to 3 and grade 2–4, respectively). By Day 28, 17 of 23 horses (74%) showed improvement in their ESGD scoring. Six horses (26%) showed no improvement at the end of the study. Refer to Figure 1 for distribution of ESGD and EGGD grades across timepoints. ESGD scores were significantly lower on Day 14 and 28 (p = 0.0035 and p = 0.0002, respectively). ESGD scores from Day 14 to 28 did not change significantly (p > 0.99). The pylorus was not visualised across all timepoints despite
the consistent fasting protocol as gastric fluid occasionally obstructed the gastroscopic view. Six horses had EGGD lesions identifiable at the initial evaluation and Day 28, and the data were not interpreted at Day 14 given the lack of data from all horses at that timepoint. Five of the six (83%) originally identified horses with EGGD were healed by Day 28. The remaining horse had an increase in EGGD score by 1 score. In the six horses evaluated, EGGD scores did not significantly change from baseline to Day 28 (p > 0.99). Three of the 23 horses in the study were concurrently
receiving daily oral firocoxib [Equioxx: Merial Inc.] at 57 mg by mouth every 24 h for the management of chronic osteoarthritis. Therapy was started at least one month prior to study start and lasted past the duration of the study. Statistical analysis was performed blocking these horses out with similar findings in changes in ESGD and EGGD grades from horses not treated with firocoxib (for Day 0 to 14 change, p = 0.0015 for ESGD and p > 0.99 for EGGD). Of the three horses receiving firocoxib treatment, two showed static ulcer grades (ESGD and EGGD), and one showed improvement in both ESGD and EGGD.
Safety Injection site reactions received higher scores following the third and fourth injections, and more horses experienced injection site reactions following multiple doses, with 8% having reactions following first dosing and 48% following fourth dosing (Figs 2 and 3). Doses three and four were performed in the side of the neck that had received an injection 2 weeks prior. All horses received one dose of
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