Prolonged administration of oral phenylbutazone and firocoxib in horses has no impact on selected cytokine and growth factor concentrations in platelet-rich plasma and autologous protein solution.

OBJECTIVE: To determine the effects of prolonged administration of the oral NSAIDs phenylbutazone and firocoxib on concentrations of cytokines and growth factors in platelet-rich plasma (PRP) and autologous protein solution (APS). ANIMALS: 6 adult University owned horses. METHODS: Horses were randomized to receive phenylbutazone (1 g, orally, q 12 h) or firocoxib (57 mg, orally, q 24 h) for 6 days. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before the administration of NSAIDs and at 7 days (1 day following cessation of NSAIDs). Horses underwent a two-week washout period, during which blood was obtained at 14 days and 21 days. The protocol was repeated with a crossover design. PRP and APS were analyzed for concentrations of platelets, leukocytes, and several cytokines (IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) and growth factors (PDGF, FGF-2, and TGF-ß1) using immunoassays. Plasma was evaluated for drug concentrations. RESULTS: No significant differences existed in concentrations of growth factors and cytokines before or after prolonged administration of NSAIDs. There were significant differences in concentrations of leukocytes and platelets in PRP compared to APS, with higher concentrations of leukocytes at the day 7 time point (T) in APS (phenylbutazone) and in concentrations of platelets in APS at T0 (firocoxib) and in APS at T7 (phenylbutazone). CLINICAL RELEVANCE: Veterinarians can recommend the administration of these oral NSAIDs prior to obtaining blood for PRP and APS provided a single-day washout period is instituted.

Simultaneous quantification and confirmation of oxycodone and its metabolites in equine urine using ultra-high performance liquid chromatography-tandem mass spectrometry.

Oxycodone, an opioid commonly used to treat pain in humans, has the potential to be abused in racehorses to enhance their performance. To understand the pharmacokinetics of oxycodone and its metabolites in horses, as well as to detect the illegal use of oxycodone in racehorses, a method for quantification and confirmation of oxycodone and its metabolites is needed. In this study, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that can simultaneously quantify and confirm oxycodone and eight metabolites in equine urine. Samples were subjected to enzymatic hydrolysis and then liquid-liquid extraction using ethyl acetate. The analyte separation was achieved on a Hypersil Gold C18 sub-2 µm column and analytes were detected on a triple quadrupole mass spectrometer. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 25-50 pg/mL and 100 pg/mL, respectively. Excellent linearity of the calibration curves was observed over a range of 100-10000 pg/mL for all nine analytes. Retention time, signal-to-noise ratio, and product ion ratios were utilized as confirmation criteria, with the limits of confirmation (LOC) ranging from 100 to 250 pg/mL. The data from a pilot pharmacokinetic (PK) study suggested that oxycodone metabolites have longer detection periods in equine urine compared to oxycodone itself; thus, the detection of metabolites in equine urine extends the ability to detect oxycodone exposure in racehorses.

Single-dose nonsteroidal anti-inflammatory drugs in horses have no impact on concentrations of cytokines or growth factors in autologous protein solution and platelet-rich plasma.

OBJECTIVE: To determine the effects of a single dose of the NSAIDs phenylbutazone, firocoxib, flunixin meglumine, and ketoprofen on concentrations of growth factors and cytokines in autologous protein solution (APS) and platelet-rich plasma (PRP). ANIMALS: 6 adult university-owned horses. METHODS: For the first phase, 6 horses were randomized to receive ketoprofen (1,000 mg) or flunixin meglumine (500 mg) IV. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before and 6 hours after administration of NSAIDs. Horses underwent a 2-week washout period, after which the protocol was repeated using a crossover design. For the second phase, following at least a 2-week washout period, the study protocol was repeated with phenylbutazone (1 g) or firocoxib (57 mg) administered orally. Plasma was collected 6 hours after administration for evaluation of drug concentrations, and APS and PRP were analyzed for concentrations of drug, platelets, leukocytes, and several growth factors and cytokines (PDGF, fibroblast growth factor, TGF-ß1, IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) before and 6 hours after administration of NSAIDs using immunoassays. RESULTS: There were no significant differences in concentrations of cytokines or growth factors before or after administration of any NSAID. There were significant differences in concentrations of leukocytes and platelets based on both product and time. NSAID concentrations in plasma were not significantly different from concentrations in APS and PRP. CLINICAL RELEVANCE: These results help guide clinicians on the appropriate use of these NSAIDs in conjunction with the processing of APS and PRP, which is unlikely to significantly alter the final product after single-dose administration.

Measurement of 8-hydroxy-2'-deoxyguanosine in serum and cerebrospinal fluid of horses with neuroaxonal degeneration and other causes of proprioceptive ataxia.

BACKGROUND: Eight-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative damage evaluated in human neurodegenerative disease, has potential to correlate with postmortem diagnosis of neuroaxonal dystrophy/degenerative myeloencephalopathy (NAD/DM) in horses. HYPOTHESIS: We hypothesized that 8-OHdG will be higher in CSF and serum from NAD/DM horses compared with horses with other neurologic diseases (CVSM, EPM) and a control group of neurologically normal horses. We also hypothesized that 8-OHdG will be higher in CSF compared with serum from NAD/DM horses. ANIMALS: Fifty client-owned horses with postmortem diagnoses: 20 NAD/DM, 10 CVSM, 10 EPM, and 10 control horses. Serum and CSF samples were obtained between November 2010 and March 2022. METHODS: Case-control study using biobanked samples was performed and commercial competitive ELISA kit (Highly Sensitive 8-OHdG Check ELISA) utilized. Concentration of 8-OHdG was quantitated in both CSF and serum and compared between groups. RESULTS: No correlation was established between the measures of 8-OHdG in serum and CSF and group. CSF median [8-OHdG] for NAD/DM was 169.9 pg/mL (IQR25-75 : 67.18-210.6), CVSM 157.1 pg/mL (IQR25-75 : 132.1-229.1), EPM 131.4 pg/mL (IQR25-75 : 102.1-193.2), and control 149.8 pg/mL (IQR25-75 : 113.3-196.4). Serum median [8-OHdG] for NAD/DM was 130 pg/mL (IQR25-75 : 51.73-157.2), CVSM 125.8 pg/mL (IQR25-75 : 62.8-170.8), EPM 120.6 pg/mL (IQR25-75 : 87.23-229.7), and control 157.6 pg/mL (IQR25-75 : 97.15-245.6). Poisson regression analysis showed no difference established once confounding variables were considered. CONCLUSIONS: Eight-OHdG did not aid in antemortem diagnosis of NAD/DM in this cohort of horses. At the time of diagnosis horses with NAD/DM do not have ongoing oxidative stress.

School psychologists' perceptions of the effectiveness of neuropsychological evaluation reports.

Referral sources and parents value the report following a neuropsychological evaluation. Nevertheless, key stakeholders have described pediatric reports as excessive in length and jargon. Recent research indicates that it is possible to modify pediatric neuropsychological reports that result in positive outcomes for key stakeholders and clinicians. Evaluating modified pediatric neuropsychological reports for other providers is necessary. School psychologists are key stakeholders who read and interact with such reports. This study assessed school psychologists' perceptions of a modified pediatric neuropsychological report. Forty-one school psychologists were randomly assigned to read either a traditional or modified version of a pediatric report and provide feedback via survey and qualitative questions. Results revealed that school psychologists' perceptions of a traditional and modified report were not significantly different. Qualitatively, respondents noted a disconnect between recommendations and school systems. These findings suggest that pediatric neuropsychologists can create shorter and more easily understood reports that do not impact the effectiveness for school psychologists. Future research should continue to evaluate perceptions of modified pediatric neuropsychological reports for additional key stakeholders. A better understanding of the disconnect between recommendations and their feasibility in schools, as well as barriers to increased interdisciplinary collaboration, is also essential for client care.

Is there an outcome benefit? Patient engagement technology in addition to the electronic medical record patient portal following elective colorectal surgery.

BACKGROUND: Patient engagement technologies (PET) are an area of growing innovation and investment, but whether PET use in the setting of electronic medical record (EMR) supported patient portals are associated with improved outcomes is unknown. Therefore, we assessed PET and EMR activation among patients undergoing elective colorectal surgery on an enhanced recovery pathway. METHODS: We identified adults undergoing elective colorectal surgery between 1/2017 and 7/2021. EMR activations were assessed and patients were considered PET users if they used a proprietary PET application. Multivariable logistic regression was used to identify factors associated with PET use and determine whether the level of engagement (percentage of messages read by the patient) was associated with 30-day outcomes. RESULTS: 484 patients (53.5% PET users, 81.6% with an activated EMR patient portal, 30.8% ≥ 70 years of age) were included. PET users were younger, more likely to have their EMR portal activated and had decreased odds of prolonged length of stay [odds ratio (OR) 0.5, 95% confidence interval (CI) 0.4-0.8]. Among patients ≥ 70 years, PET users had reduced odds of readmissions (OR 0.2, 95% CI 0.1-0.9) compared to PET non-users. The most engaged PET users had decreased morbidity (OR 0.2, 95% CI 0.1-0.8) and readmissions (OR 0.3, 95% CI 0.1-0.8) compared to the least engaged PET users. CONCLUSION: When controlling for EMR activation, patients who use PET, specifically those with higher levels of engagement or aged ≥ 70, have improved outcomes following elective colorectal surgery. Interventions aimed at increasing the adoption of PET among older adults may be warranted.

Pharmacokinetics and pharmacodynamics of intranasal and intramuscular administration of naloxone in working dogs administered fentanyl.

BACKGROUND: Working dogs exposed to narcotics might require reversal in the field. OBJECTIVE: To explore the pharmacokinetic and pharmacodynamic effects of naloxone administered intramuscularly (IM) or intranasally (IN) to reverse fentanyl sedation in working dogs. ANIMALS: Ten healthy, working dogs aged 1.7 ± 1 year and weighing 26 ± 3 kg. METHODS: In this randomized, controlled cross-over study dogs received either 4 mg of naloxone IN or IM 10 minutes after fentanyl (0.3 mg IV) administration. Sedation was assessed at baseline and 5 minutes after fentanyl administration, then at 5, 10, 15, 20, 25, 30, 60 and 120 minutes after reversal with naloxone. Blood samples for naloxone detection were obtained at 0, 5, 10, 30, 60 and 120 minutes. Pharmacokinetic parameters and sedation scores were compared between IM and IN naloxone groups. RESULTS: There was a significant increase in sedation score from baseline (0.25 [-4 to 1] IM; 0 [-2 to 1] IN) after fentanyl administration (11 [5-12] IM; 9.25 [4-11] IN), followed by a significant reduction at 5 (0.5 [-0.5 to 1.5] IM; 1.25 [-1.5 to 4.5] IN) through 120 minutes (-0.5 [-2 to 1] IM; 0 [-4.5 to 1] IN) after reversal with naloxone. Route of administration had no significant effect on sedation score. Maximum plasma concentration was significantly lower after IN administration (11.7 [2.8-18.8] ng/mL IN, 36.7 [22.1-56.4] ng/mL IM, P < .001) but time to reach maximum plasma concentration was not significantly different from IM administration. CONCLUSION AND CLINICAL IMPORTANCE: Although IM administration resulted in higher naloxone plasma concentrations compared to IN, reversal of sedation was achieved via both routes after administration of therapeutic doses of fentanyl.

Factors affecting untargeted detection of doping agents in biological samples.

Doping control is essential for sports, and untargeted detection of doping agents (UDDA) is the holy grail for anti-doping strategies. The present study examined major factors impacting UDDA with metabolomic data processing, including the use of blank samples, signal-to-noise ratio thresholds, and the minimum chromatographic peak intensity. Contrary to data processing in metabolomics studies, both blank sample use (either blank solvent or plasma) and marking of background compounds were found to be unnecessary for UDDA in biological samples, the first such report to the authors' knowledge. The minimum peak intensity required to detect chromatographic peaks affected the limit of detection (LOD) and data processing time for untargeted detection of 57 drugs spiked into equine plasma. The ratio of the mean (ROM) of the extracted ion chromatographic peak area of a compound in the sample group (SG) to that in the control group (CG) impacted its LOD, and a small ROM value such as 2 is recommended for UDDA. Mathematical modeling of the required signal-to-noise ratio (S/N) for UDDA provided insights into the effect of the number of samples in the SG, the number of positive samples, and the ROM on the required S/N, highlighting the power of mathematics in addressing issues in analytical chemistry. The UDDA method was validated by its successful identification of untargeted doping agents in real-world post-competition equine plasma samples. This advancement in UDDA methodology will be a useful addition to the arsenal of approaches used to combat doping in sports.

Effect of sirolimus on insulin dynamics in horses.

BACKGROUND: Sirolimus, a mechanistic target of rapamycin inhibitor, suppresses insulin production in other species and has therapeutic potential for hyperinsulinemia in horses. HYPOTHESIS/OBJECTIVE: Determine the pharmacokinetics (PKs) of sirolimus and evaluate its effect on insulin dynamics in healthy and insulin dysregulation (ID) horses. ANIMALS: Eight Standardbred geldings. METHODS: A PK study was performed followed by a placebo-controlled, randomized, crossover study. Blood sirolimus concentrations were measured by liquid chromatography-mass-spectrometry. PK indices were estimated by fitting a 2-compartment model using nonlinear least squares regression. An oral glucose test (OGT) was conducted before and 4, 24, 72, and 144 hours after administration of sirolimus or placebo. Effects of time, treatment and animal on blood glucose and insulin concentrations were analyzed using mixed-effects linear regression. Sirolimus was then administered to 4 horses with dexamethasone-induced ID and an OGT was performed at baseline, after ID induction and after 7 days of treatment. RESULTS: Median (range) maximum sirolimus concentration was 277.0 (247.5-316.06) ng/mL at 5 (5-10) min and half-life was 3552 (3248-4767) min. Mean (range) oral bioavailability was 9.5 (6.8-12.4)%. Sirolimus had a significant effect on insulin concentration 24 hours after a single dose: median (interquartile range) insulin at 60 min (5.0 [3.7-7.0] µIU/mL) was 37 (-5 to 54)% less than placebo (8.7 [5.8-13.7] µIU/mL, P = .03); and at 120 min (10.2 [8.4-12.2] µIU/mL) was 28 (-15 to 53)% less than placebo (14.9 [8.4-24.8] µIU/mL, P = .02). There was minimal effect on glucose concentration. Insulin responses decreased toward baseline in ID horses after 7 days of treatment. CONCLUSION AND CLINICAL IMPORTANCE: Sirolimus decreased the insulinemic response to glucose and warrants further investigation.

Use of Liquid Chromatography--Tandem Mass Spectrometry to Quantify and Confirm the Fentanyl Metabolite N-[1-(2-Phenethy-4-Piperidinyl)] Maloanilinic Acid in Equine Urine for Doping Control.

Fentanyl, a powerful synthetic mu opioid receptor agonist, is banned in equine sports by the Association of Racing Commissioners International and the Fédération Équestre Internationale. The presence of fentanyl in equine blood has been confirmed during routine post-race screening for doping substances in the authors' laboratory. While fentanyl can be detected and confirmed in blood, it is rapidly metabolized, and screening for the metabolite N-[1-(2-phenethy-4-piperidinyl)] maloanilinic acid (PMA) in equine urine is expected to allow for a longer detection time. In this study, a quantitative and confirmatory liquid chromatography--tandem mass spectrometry (LC-MS-MS) method was developed for PMA analysis in equine urine. PMA was extracted by solid phase extraction, separated on a C18 column and detected using a triple quadrupole mass spectrometer. The mass spectrometer was operated in positive-ion mode, and multiple reaction monitoring was used to monitor product ions m/z 188, m/z 281 and m/z 323. The method was validated for extraction recovery, matrix effect, specificity, sensitivity, precision and accuracy, carryover and processed sample stability according to the guidelines of the US Food and Drug Administration for bioanalysis. The limits of detection and quantification were 5 and 10 pg/mL, respectively. Linearity was obtained over the concentration range of 10-10,000 pg/mL. To confirm PMA in equine urine, LC retention time, diagnostic product ions (m/z 188, m/z 281 and m/z 323) and product ion ratio were used as the criteria. The lowest concentration for confirmatory analysis was validated at 50 pg/mL. The method was applied to measure the PMA concentrations in equine urine following intravenous administration of fentanyl to a research horse and has confirmed the presence of PMA in post-race urine samples. This method is a valuable addition to the arsenal of equine doping control methods to combat illegal doping and protect racehorse health.

Automated identification of unknown doping agents in confiscation samples by flow-injection mass spectrometry and mass spectral library searches.

Rapid and accurate identification of unknown compounds within suspicious samples confiscated for sports doping control and law enforcement drug testing is critical, but such analyses are often conducted manually and can be time-consuming. Here, we report a methodology for automated identification of unknown substances in confiscation samples by rapid automatic flow-injection analysis on a liquid chromatography coupled to high-resolution mass spectrometry system and identifying unknown compounds with Compound Discoverer software. The developed methodology was validated by comparing the automated identification results with those obtained from manual syringe-infusion experiments and manual tandem mass spectral library searches. The automated methodology resulted in far higher throughput and remarkably shorter turnaround time for analysis when compared with manual procedures and, in most cases, yielded more compounds. As this is the first such report to the authors' knowledge, this methodology may potentially transform analysis of confiscated samples in sports doping control and law enforcement drug testing.

Administration and detection of gene therapy in horses: A systematic review.

Gene therapy uses genetic modification of cells to produce a therapeutic effect. Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression. Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses. Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.

Prerace venous blood gases and acid-base values in Standardbred horses: effects of geography, season, prerace furosemide, gender, age, and trainer using big data analytics.

OBJECTIVE: A retrospective study was conducted to establish the prerace venous acid-base and blood gas values of Standardbred horses at rest using big data analytics. SAMPLES: Venous blood samples (73,382) were collected during seven racing seasons from 3 regional tracks in the Commonwealth of Pennsylvania. Horses were detained 2 hours prior to race time. PROCEDURES: A mixed-effects linear regression model was used for estimating the marginal model adjusted mean (marginal mean) for all major outcomes. The interaction between age and gender, track, and the interaction between month, treatment (furosemide), and year were the major confounders included in the model. Random effects were set on individual animal nested within trainer. Partial pressure of venous carbon dioxide (PVCO2), partial pressure of oxygen (PVO2), and pH were measured, and base excess (BE), total carbon dioxide (TCO2), and bicarbonate (HCO3-) were calculated. RESULTS: Significant (P < .001) geographical differences in track locations were seen. Seasonal reductions in acid-base values started in January with significant (P < .001) decreases from adjacent months seen in June, July, and August followed by a gradual return. There were significant increases (P < .001) in BE and TCO2 and decreases in PVO2 with age. Significant differences (P < .001) in acid-base values were seen when comparing genders. A population of trainers were significantly different (P < .001) from the marginal mean and considered outliers. CLINICAL RELEVANCE: In a population of horses, big data analytics was used to confirm the effects of geography, season, prerace furosemide, gender, age, and trainer influence on blood gases and the acid-base profile.

Effect of a GLP-1 mimetic on the insulin response to oral sugar testing in horses.

BACKGROUND: Insulin dysregulation (ID) is the most important risk factor for the development of laminitis in horses and therapies to control it are needed. HYPOTHESIS/OBJECTIVES: To assess the effects of a single dose of the synthetic GLP-1 analog exenatide on postprandial insulin dynamics. We hypothesized that exenatide would improve insulin sensitivity and lower postprandial blood insulin concentrations. STUDY DESIGN: Randomized, crossover, experimental study. ANIMALS: Six horses (3 mares, 3 geldings; 2 with normal insulin regulation [NIR] and 4 with mild ID). METHODS: Horses completed both study arms: subcutaneous administration of exenatide (or no treatment) 30 min before an oral sugar test (0.15 ml/kg of Karo Syrup). Blood samples obtained over 240 min were assayed for glucose, insulin, lactate, c-peptide and total GLP-1. The area under the curve (AUC) was calculated using the trapezoidal rule. Insulin sensitivity (SI) was estimated using a mathematical model. RESULTS: Exenatide resulted in a postprandial decrease of 20% (effect size: 2673 µU·min/ml; 95% CI: 900 - 4446 µU·min/ml; P = 0.003) in AUC of plasma insulin (control; mean AUC insulin: 11,989 µU·min/ml; 95% CI: 9673 - 14,305 µU·min/ml, exenatide; mean AUC insulin: 9316 µU·min/ml; 95% CI: 7430 - 11,202 µU·min/ml). Exenatide resulted in an approximately threefold increase (effect size: 5.56 10-4· µU/ml-1·min-1; 95% CI: 0.95 - 10.1 10-4· µU/ml-1·min-1; P = 0.02) in estimated insulin sensitivity (control mean SI: 1.93 10-4· µU/ml-1·min-1; 95% CI: 0.005 - 3.86 10-4·µU/ml-1·min-1 vs. exenatide mean SI: 7.49 10-4· µU/ml-1·min-1; 95% CI: 3.46 - 11.52 10-4· µU/ml-1·min-1). CONCLUSIONS: The decrease in insulin response to carbohydrates was due to an increase in whole-body insulin sensitivity. GLP-1 agonists may have therapeutic potential for ID in horses.

Pharmacokinetics of glaucine after intravenous and oral administrations and detection of systemic aporphine alkaloids after ingestion of tulip poplar shavings in horses.

Glaucine, an aporphine alkaloid with anti-tussive, anti-inflammatory, and anti-nociceptive properties, has been identified in post-race samples from racehorses. To investigate pharmacokinetics of glaucine in horses, a three-way crossover study of intravenous and oral glaucine (0.1 mg/kg) and orally administered tulip poplar shavings (50 g shavings = 0.001 mg/kg glaucine) was performed in six horses. A two-compartment model best described IV administration with alpha ( t 1 / 2 α ) and beta ( t 1 / 2 ß ) half-life lives of 0.3 (0.1-0.7) and 3.1 (2.4-7.8) h, respectively. The area under the curve ( AUC 0 ∞ iv ) was 45.4 (34.7-52.3) h*ng/ml, and the volume of distribution of the central (Vdc ) and peripheral (Vdp ) compartments was 2.7 (1.3-4.6) and 4.9 (4.3-8.2) L/kg, respectively. A one compartment model best described the oral administration of glaucine with absorption ( t 1 / 2 ka ) and elimination ( t 1 / 2 kel ) half-lives of 0.09 (0.05-0.15) and 0.7 (0.6-0.8) h, respectively. The area under the curve ( AUC 0 ∞ PO ) was 15.1 (8.0-19.5) h·ng/ml. Bioavailability following oral administration was 17%-48%. Following ingestion of shavings, glaucine and liriodenine were detectable in plasma for up to 16 and 48 h, respectively. Glaucine was quantifiable briefly in the urine from two horses. Liriodenine was quantifiable in urine for 12-20 h in four horses and for 48 h in two horses. The presence of liriodenine indicates ingestion of tulip poplar tree parts, however, does not rule out co-administration of purified glaucine in horses.

Critically important antimicrobials are frequently used on equine racetracks.

OBJECTIVE: To characterize antimicrobial use on four racetracks in the eastern US during the peak racing 2017-2018 seasons. PROCEDURES: Handwritten daily treatment sheets provided by attending veterinarians who listed treatments administered to horses stabled at the racetrack were obtained. Information contained in the treatment sheets included the date, name of the horse and its trainer, type of treatment, and a brief (usually 1-word) indication for treatment. The handwritten data listed on the racetrack treatment sheets were manually transcribed and analyzed. RESULTS: A total of 2,684 antimicrobial prescriptions were recorded, representing 6.8% of all drug treatments. The most frequently prescribed antimicrobials were enrofloxacin, with 854 prescriptions (31.8% of antimicrobial treatments), followed by gentamicin (570 [21.2%] prescriptions), ceftiofur (388 [14.5%] prescriptions,), and penicillin (220 [8.2%] prescriptions). The relative frequencies of antimicrobial class and indication for treatment varied significantly by racetrack and by prescribing veterinarian. Limitations associated with the data precluded ascertainment of the proportion of horses treated or exact indications for treatment. CLINICAL RELEVANCE: Antimicrobials appeared to be prescribed relatively infrequently at racetracks relative to other drugs, but highly or critically important antimicrobials were most often used. The appropriateness of use of these drugs remains unknown.

Gene transcripts expressed in equine white blood cells are potential biomarkers of extracorporeal shock wave therapy.

Extracorporeal shockwave therapy (ESWT) is a treatment applied to musculoskeletal injuries in equine athletes to alleviate pain and accelerate healing. ESWT also causes acute tissue damage. Therefore, its ability to act as an analgesic and cause tissue damage potentially increases the risk of a catastrophic event if used shortly before a strenuous competition such as horseracing. While ESWT is prohibited by many racing jurisdictions within 10 days prior to competition, a test to detect whether a horse has received ESWT is needed. ESWT changes the protein levels of inflammatory mediators in blood, and white blood cells (WBC) typically produce these proteins. Changes in gene expression precede changes in protein production; thus, it was hypothesized that WBC gene transcripts might serve as biomarkers of ESWT. To test this hypothesis, six thoroughbred horses received a single administration of ESWT to the distal limb, and WBC RNA was extracted from blood samples collected before (0 h) and after ESWT (2, 4, 6, 24, 48, and 72 h). Targeted and untargeted analyses evaluated the transcriptome using quantitative PCR (qPCR) and microarray. The expression of IL-1α, IL-1ß, TNF-α, IL-1Ra1, IL-1Ra2 and TGF-ß1, and BMPR1A in circulating WBCs was significantly up-regulated, while IFN-γ, ZNF483, TMEM80, CAH6, ENPP, and S8723 were significantly down-regulated at various time points following ESWT. These data support the hypothesis that changes in WBC gene transcripts could serve as biomarkers for ESWT.

A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping.

Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequences to enable theoretical detection of all rAAV gene therapy products, regardless of encoded transgenes while excluding the presence of WTAAV DNA in horses. Primer sets were developed and validated to target an AAV2 sequence highly conserved across rAAV viral vectors and a sequence only found in wild type AAV2 (WTAAV2). Six horses were administered an intra-articular injection of rAAV. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Using QPCR, rAAV was detected in plasma for up to 2-4 days in all horses. rAAV DNA was detected for 28 days in synovial fluid from two horses for which synovial fluid samples were available. No WTAAV2 DNA was detected in any sample. This is the first study to develop a QPCR test capable of screening for rAAV vectors that may be used for gene doping in horses.

Pharmacokinetics and pharmacodynamics of oral and intravenous metoprolol tartrate in clinically healthy horses.

Cardiac drugs with defined pharmacological parameters in horses are limited. The objective of this study was to characterize the pharmacokinetic properties and cardiovascular effects of intravenous and oral metoprolol tartrate (MET) in horses. In a 2-period randomized cross-over design, MET was administered IV (0.04 mg/kg) and PO (6 mg/kg) once to six healthy adult horses. Horses were monitored via continuous telemetry and non-invasive blood pressure (NIBP). Blood samples were serially collected for 72 h post-administration, and concentrations were determined by LC-MS/MS. Pharmacokinetics were modeled using a 3-compartment model and non-linear least squares regression. Median (range) MET concentration was 110 (40.1-197) ng/ml collected 1 min (0.0167 h) after a bolus IV administration. Maximum concentration (Cmax ) after PO administration was 2135 (1590-4170) ng/ml at 0.5 (0.25-0.5) hours. Oral bioavailability was 54% (17-100%). Median apparent volume of distribution was 0.39 (0.17-0.58) l/kg, clearance was 12.63 (11.41-18.94) ml/kg/min, and elimination half-life was 21.1 (7.46-34.36) minutes. No clinically relevant effects of IV or PO metoprolol were noted on cardiac rhythm or NIBP. Sweating was the most common side effect. The metoprolol doses used in this study achieve plasma concentrations reported to achieve ß-blockade in humans.

Novel Algorithms for Comprehensive Untargeted Detection of Doping Agents in Biological Samples.

To address the limitations of current targeted analytical methods that can only detect known doping agents, a novel methodology that permits untargeted drug detection (UDD) has been developed to help in the fight against doping in sports. Fifty-seven drugs were spiked into blank equine plasma and were treated as unknowns since their exact masses and chromatographic retention times were not utilized for detection. The spiked drugs were extracted from the plasma samples and were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). The acquired LC-HRMS raw data files were processed using metabolomic software for compound detection and identification. For UDD with the resultant data, a mathematical model was created, and two algorithms were generated to calculate the ratio of the mean (ROM) and outlier index (OLI). Using ROM and OLI, the majority of the 57 drugs were accurately detected by name (52 of 57) or chemical formula (1 of 57). The limit of detection for the drugs was from tens of picograms to nanograms per milliliter. Xenobiotics and endogenous substances relevant to doping control were also identified using this untargeted approach following their extraction from real-world race samples, thus validating the UDD methodology. To the authors' knowledge, this is the first completely UDD methodological approach and represents significant advance toward using artificial intelligence for the detection of both known and emerging doping agents in sports.