Pharmacokinetic study of osilodrostat and identification of mono-hydroxylated metabolite in equine plasma for the purpose of doping control.

RATIONALE: Osilodrostat is an inhibitor of 11-beta-hydroxylase (CYP11B) and is used for the treatment of Cushing's disease but also categorized as an anabolic agent. The use of osilodrostat is prohibited in horseracing and equestrian sports. To the best of our knowledge, this is the first metabolic study of osilodrostat in equine plasma. METHODS: Potential metabolites of osilodrostat were identified by differential analysis using data acquired from pre- and post-administration plasma samples after protein precipitation with liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS). [Correction added on 27 January 2023, after first online publication: In the preceding sentence, "C-HRMS" was changed to "LC/ESI-HRMS" in this version.] For quantification of osilodrostat, a strong cation exchange solid-phase extraction was employed, and the extracts were analyzed using LC/ESI-triple quadrupole tandem mass spectrometry (LC/ESI-QqQ-MS/MS) to establish its elimination profile. Such extracts were further analyzed using LC/ESI-HRMS to investigate the detectability of osilodrostat and its identified mono-hydroxylated metabolite over a 2-week sampling period. RESULTS: Mono-hydroxylated osilodrostat was identified based on the differential analysis and mass spectrometric interpretations, and it was found to be the most abundant metabolite in plasma. Elimination profile of osilodrostat in plasma was successfully established over the 24-h post-administration period. Both osilodrostat and its mono-hydroxylated metabolite were detected up to the last sampling point at 2 weeks using HRMS, and osilodrostat could be confirmed up to 8-day post-administration with its reference material using HRMS as well. CONCLUSIONS: For doping control, screening of both the parent drug osilodrostat and its mono-hydroxylated metabolite in equine plasma would be recommended due to their extended detection windows of up to 2 weeks. Given the availability of reference material for potential confirmation in forensic samples, osilodrostat is considered the most appropriate monitoring target.

Evaluation of plasma and urine pharmacokinetics of tranexamic acid for equine medication control.

This study aimed to evaluate the pharmacokinetics (PK) of tranexamic acid (TXA) in horses and estimate its irrelevant plasma and urine concentrations using the pharmacokinetic/pharmacodynamic (PK/PD) approach by applying the Pierre-Louis Toutain model. TXA was intravenously administered to eight thoroughbred mares, and plasma and urine TXA concentrations were quantified by liquid chromatography/tandem mass spectrometry. The quantified data were used to calculate the PK parameters of TXA in horses. The plasma elimination curves were best-fitted to a three-compartment model. Using the Toutain model approach, irrelevant plasma and urine TXA concentrations were estimated to be 0.0206 and 0.997 µg/mL, respectively. The typical values of clearance, steady-state volume of distribution, and steady-state urine-to-plasma ratio were 0.080 L/kg/h, 0.86 L/kg, and 49.0, respectively. The obtained irrelevant concentrations will be useful for establishing relevant regulatory screening limits for effective control of TXA use in horse racing and equestrian sports.

Heritability estimates of fractures in Japanese Thoroughbred racehorses using a non-linear model.

Thoroughbred racehorses are produced by mating small numbers of Arabian stallions and native British mares, and have been improved by selection of horseracing performance for about 300 years. While these improvements led to good performance as racehorses, they exposed horses to numerous medical disorders, aggravated by extensive exercise. Fractures are frequent medical disorders in Thoroughbred racehorses. In this study, fracture heritability was estimated using 3,927 Japanese Thoroughbred racehorses to elucidate the risk of racehorse fractures. The heritability estimates of all examined fractures were low (h2  = 0.06), while those of fractures in carpal bone and carpus (carpal bone plus distal radius) were moderate (h2  = 0.37, 0.24, respectively). Fracture occurrence age for carpal bone and distal radius was both 3.3 years old and was younger than that for other fractures. These results indicated that a larger proportion of the variation in the studied population was due to genetic factors for carpal fractures than for other fractures, while the fractures at other bones were largely affected by environmental factors, correlated with the athlete period (number year in racing). These findings contribute to develop a management plan for suppressing racehorse fractures and improving horseracing safety.

Prevalence of post-race exertional heat illness in Thoroughbred racehorses and climate conditions at racecourses in Japan.

Despite growing recognition of post-race exertional heat illness (EHI) in the horse racing industry, reports on its prevalence are limited. The purpose of this study was to investigate the prevalence of post-race EHI and climate conditions at racecourses in Japan. The overall prevalence of EHI from 1999 to 2018 was 0.04% (387 cases for 975,247 starters) in races operated by the Japan Racing Association (JRA). The yearly prevalence has been increasing, exceeding 0.07% in the last four years of the studied period. The overall prevalence in summer (May-September) was 0.086% (352 cases for 409,908 starters). The monthly prevalence varied among the 10 JRA racecourses, which are distributed from latitude 34 to 43°N, ranging from no cases to 0.459%. During summer, prevalence of post-race EHI was high when the mean monthly ambient temperature was high at a racecourse. To evaluate climate conditions, we investigated the wet-bulb globe temperature (WBGT, °C) from 9 AM to 5 PM on sunny race days in July and August of 2017 and 2018 at three racecourses with a high prevalence of EHI among the 10 racecourses. The durations of time during which WBGT was between 28 and 33°C at these three courses were 95, 94, and 65% of the minutes measured, respectively. This result indicated that most races on the sunny summer days were held when WBGT was between 28 and 33°C at the three racecourses. These findings could be useful in developing the appropriate countermeasures to be taken during hot weather at each of the studied racecourses.

Evaluating continuous blood coagulopathy in assessing the severity of acute colitis in Thoroughbred racehorses.

Although severe blood coagulopathy in horses with acute colitis causes multiple organ failure, which may be fatal, few studies have focused on the correlation between the fluctuations of coagulation parameters and severity of colitis. In this study, we evaluated the fluctuations of coagulation parameters in 14 Thoroughbred racehorses with acute colitis for 5 days from the day of hospitalization and compared them between 5 survivors and 9 non-survivors. Noteworthy features in the non-survivors were that antithrombin activity and fibrin degradation products continuously decreased and increased, respectively, for 4 days or more and that thrombin-antithrombin complexes increased in the last 2 days before death. Thus, these parameters should be continuously monitored to observe these fluctuations in assessing the severity of acute colitis.

Blood glucose is unlikely to be a prognostic biomarker in acute colitis with systemic inflammatory response syndrome in Thoroughbred racehorses.

Although hyperglycemia at admission with colic has been reported to have a poor prognosis, there is no report specifically about acute colitis with systemic inflammatory response syndrome (SIRS) in horses. In this study, we measured blood glucose (Glu), insulin (Ins), and cortisol (Cor) levels in 17 Thoroughbred racehorses diagnosed as having acute colitis with SIRS, and examined the relationship between time-dependent changes in Glu, Ins, and Cor and prognosis. Glu levels were high in 3 horses at admission, but thereafter no horses had persistently high Glu levels. There was no significant difference in Glu, Ins, and Cor levels within 72 hr between surviving and non-surviving horses. In conclusion, the Glu level is unlikely to be a useful prognostic biomarker in acute colitis with SIRS.

Signal changes in standing magnetic resonance imaging of osseous injury at the origin of the suspensory ligament in four Thoroughbred racehorses under tiludronic acid treatment.

Problems associated with the proximal metacarpal region, such as an osseous injury associated with tearing of Sharpey's fibers or an avulsion fracture of the origin of the suspensory ligament (OISL), are important causes of lameness in racehorses. In the present study, four Thoroughbred racehorses (age range, 2-4 years) were diagnosed as having forelimb OISL and assessed over time by using standing magnetic resonance imaging (sMRI). At the first sMRI examination, all horses had 3 characteristic findings, including low signal intensity within the trabecular bone of the third metacarpus on T1-weighted images, intermediate-to-high signal intensity surrounded by a hypointense rim on T2*-weighted images, and high signal intensity on fat-suppressed images. Following the sMRI examination, all horses received 50 mg of tiludronic acid by intravenous regional limb perfusion once weekly for 3 weeks. Attenuation of the high signal intensity on T2*-weighted and fat-suppressed images was observed on follow-up sMRI in 3 horses. Following rest and rehabilitation, these 3 horses successfully returned to racing. In contrast, the other horse that did not show attenuation of the high signal intensity failed to return to racing. To our knowledge, this is the first report of OISL in Thoroughbred racehorses assessed over time by sMRI under tiludronic acid treatment. Our findings support the use of sMRI for examining lameness originating from the proximal metacarpal region to refine the timing of returning to exercise based on follow-up examinations during the recuperation period.

Risk Factors for Epistaxis in Thoroughbred Flat Races in Japan (2001-2020).

We investigated the risk factors for epistaxis in Japanese flat races over a 20-year period. The veterinary records of horses identified as having epistaxis by endoscopy on the race day, and the official racing records of all flat races from April to September between 2001 and 2020, were reviewed. The racecourses (n = 10), surface type, surface condition, race class, race distance, race year, sex, age, two training centers, ambient temperature, and body weight on race days were assessed using multivariable logistic regression (p < 0.05). Of 475,709 race starts, 616 (1.30 cases per 1000 starts; 95% confidence interval [CI], 1.20-1.40) included an epistaxis event. Nine variables were significantly associated with epistaxis. Seven of the variables have been reported in previous studies: lower ambient temperature, soft surface conditions, shorter racing distances (≤1400 m), increasing age, females and geldings compared to males, training center, and race year. However, two novel variables were identified as significantly associated with epistaxis, increasing body weight per 20 kg (p < 0.001, odds ratio [OR], 1.33; 95% CI, 1.25-1.41) and the racecourses that the horses were running at (p < 0.001, especially Sapporo [OR; 4.74, 95% CI, 3.07-7.31], Hakodate [OR, 4.66; 95% CI, 3.05-7.11], and Kokura [OR, 4.14; 95% CI, 2.65-6.48] compared to the reference racecourse [Kyoto]). These results can facilitate developing interventions to reduce epistaxis in flat racing.

A comprehensive and comparative proteomic analysis of horse serum proteins in colitis.

BACKGROUND: Equine colitis is a diarrhoeal disease caused by inflammation of the large bowel and can potentially be life-threatening due to its rapid progression. Pathogenesis is multifactorial and pathophysiology is highly complicated, therefore, reliable diagnostic biomarkers are needed in the veterinary field. OBJECTIVE: Serum is one of the most commonly used diagnostic tools in equine clinical investigation. To discover diagnostic or prognostic protein markers for colitis in horse serum, comprehensive and comparative proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). STUDY DESIGN: Case-control study. METHODS: Serum samples were collected from 36 healthy Thoroughbreds and 12 Thoroughbreds with colitis. Serum from each horse suffering from colitis was collected daily until death or recovery. Collected sera were digested with trypsin. Peptides obtained from serum proteins were measured by Q-Exactive HF Orbitrap mass spectrometer. The identification and quantification of peptides were performed using Proteome Discoverer version 2.2. RESULTS: On day 1 of treatment, eight proteins in the colitis group were upregulated (P < .05, more than a twofold change) compared with the healthy group. Among the eight proteins, biliverdin reductase B was significantly upregulated (P < .05) in the non-survivor group (n = 5) compared with the survivor group (n = 7). On the last day of the treatment, haemoglobin subunit alpha, clusterin, glyceraldehyde-3-phosphate dehydrogenase, lipopolysaccharide-binding protein, and biliverdin reductase B showed significant increases (P < .05) in the non-survivor group. MAIN LIMITATIONS: The number of the identified proteins is limited due to the existence of abundant proteins. CONCLUSIONS: Measuring the changes of these proteins together may enable a potential prognosis or early diagnosis of horses suffering from colitis.

Medication control of flunixin in racing horses: Possible detection times using Monte Carlo simulations.

BACKGROUND: For medication control in several jurisdictions, withdrawal time is the period of refrain from racing after drug administration. It is set by adding a safety period to an experimental detection time. However, there are no reports of statistical analyses of detection time for the determination of withdrawal time in flunixin meglumine-treated horses. OBJECTIVE: To analyse the population pharmacokinetics of flunixin in horses through the generation of a dataset for detection time statistical analysis and predictions via Monte Carlo simulation. STUDY DESIGN: Experimental study. METHODS: Drug plasma and urine concentrations following single intravenous administration of flunixin 1.1 mg/kg bodyweight (BW) in 10 horses and multiple administration of q 24 hours for 5 days in 10 horses were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Data were modelled using a nonlinear mixed effect model followed by Monte Carlo simulation. Irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were calculated using the Toutain approach. Detection times were obtained considering the time after the last administration for selected quantiles of 5000 hypothetical horses under the international screening limit (ISL) proposed by the International Federation of Horseracing Authorities (plasma: 1 ng/mL, urine; 100 ng/mL). RESULTS: For a regimen of 1.1 mg/kg BW q 24 hours, the IPC and IUC values were 2.0 and 73.0 ng/mL respectively. Detection times in plasma above the ISL for 90% of simulated horses were estimated as 74 hours after a single 1.1 mg/kg dose administration, 149 and 199 hours after multiple doses over 5 days at either 24- or 12-hour intervals respectively. Corresponding detection times in urine were 46, 68 and 104 hours respectively. MAIN LIMITATION: Only female horses were investigated. CONCLUSIONS: Statistical detection times for different flunixin meglumine regimens indicated a delay of detection time in plasma after multiple administrations under ISL.

Mortality, clinical findings, predisposing factors and treatment of Clostridioides difficile colitis in Japanese thoroughbred racehorses.

BACKGROUND: Although Clostridioides difficile-associated diseases (CDAD) is considered to be associated with colitis in horses, few studies have been performed with a focus on the characteristics of CDAD in thoroughbred racehorses. METHODS: Between 2010 and 2018, a test for C. difficile was performed using faecal samples from 137 thoroughbred racehorses with colitis presenting with diarrhoea and fever. The mortality rate, clinical findings, predisposing factors and the selected treatments were investigated in a retrospective manner. RESULTS: Twenty-four cases were diagnosed as CDAD and 113 as non-CDAD. The mortality rate was significantly higher in the CDAD group (83 per cent) than that in the non-CDAD group (34 per cent). The levels of serum amyloid A, blood urea nitrogen and packed cell volume at initial presentation were also significantly higher, and those of total protein and albumin were significantly lower in the CDAD group. The development of CDAD was associated with the administration of antimicrobials, surgery and hospitalisation. No significant improvement in mortality was observed for any of the selected treatment in both groups. CONCLUSION: CDAD in thoroughbred racehorses was identified as a high mortality disease with rapid progression of systemic inflammation and deterioration of the circulatory state. Further investigation is required to improve the treatment.

Standing magnetic resonance imaging of distal phalanx fractures in 6 cases of Thoroughbred racehorse.

Six Thoroughbred racehorses with palmar process fractures of the distal phalanx were evaluated with standing magnetic resonance imaging (sMRI). In all the cases, the fractures were detectable on T1-weighted images and fat-suppressed images. Furthermore, multi-planar reconstruction images were useful for assessing the articular involvement of the fractures. Follow-up sMRI was obtainable in 3 cases, which revealed that the area of high signal intensity on fat-suppressed images decreased over time as symptoms improved. Our findings support the use of sMRI for the detailed evaluation of distal phalanx fractures including their articular involvement and the healing process.

Bone marrow oedema-type signal in the proximal phalanx of Thoroughbred racehorses.

This study focused on 8 Thoroughbred racehorses showing bone marrow oedema-type signal in the proximal sagittal groove of the proximal phalanx, with the aim of understanding its clinical significance. Standing magnetic resonance imaging played an important role in assessing osseous abnormalities that were not radiographically identifiable. Further, a histopathological result from one of the cases showed there was oedema surrounding adipose tissues with increase in density of trabecular scaffolding. This may indicate presence of osseous injury within the area of decreased elasticity due to subchondral bone modeling. This study suggests that detection of osseous abnormality based on bone marrow oedema-type signal, and application of appropriate care following injury would contribute to prevent deterioration of stress-related fractures of the proximal phalanx.

Use of standing low-field magnetic resonance imaging to assess oblique distal sesamoidean ligament desmitis in three Thoroughbred racehorses.

Desmitis of the oblique distal sesamoidean ligaments (ODSL) is caused by hyperextension of the metacarpophalangeal/metatarsophalangeal joint and has been described as a significant cause of lameness in racehorses. In this study, three Thoroughbred racehorses (age range: 3-6 years) were diagnosed with desmitis of the forelimb ODSL using standing low-field magnetic resonance imaging (sMRI). Radiography and ultrasonography were inconclusive with regard to a definitive diagnosis. For all horses, the sMRI characteristics included increased signal intensity within the medial ODSL on T1-weighted gradient echo, T2-weighted fast spin echo and short tau inversion recovery fast spin echo images, which use a fat suppression technique. Effusion of the digital flexor tendon sheath was also clearly visible on sMRI. Following rest and controlled exercise for roughly 3 months, two horses successfully returned to racing within 5 months. Our findings support the use of sMRI for diagnosing ODSL injuries in Thoroughbred racehorses.