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.

Antimicrobial resistance profiles and phylogenetic groups of Escherichia coli isolated from healthy Thoroughbred racehorses in Japan.

In this study, we investigated the occurrence of antimicrobial resistance in commensal Escherichia coli isolated from healthy Thoroughbred (TB) racehorses in Japan. A total of 212 fecal samples were individually collected from TB racehorses from March 2017 to August 2018 at Japan Racing Association training centers. E. coli was isolated by using selective agar media, deoxycholate-hydrogen sulfide-lactose (DHL) and eosin methylene blue (EMB). A total of 417 E. coli isolates were examined against 10 antimicrobial agents by using the broth microdilution method. The 417 E. coli isolates were phylogenetically grouped using a multiplex polymerase chain reaction. The highest proportion of resistance was observed for streptomycin (30.9%, 129/417) followed by ampicillin (19.4%, 81/417), trimethoprim (15.8%, 66/417), tetracycline (8.4%, 35/417), chloramphenicol (2.6%, 11/417), kanamycin (1.2%, 5/417), nalidixic acid (0.5%, 2/417), cefazolin (0.2%, 1/417), colistin (0.2%, 1/417), and gentamycin (0%). Multidrug-resistant (MDR) E. coli was detected in 7.9% (33/417) of isolates. The proportions of resistance against ampicillin, streptomycin, kanamycin, and chloramphenicol and of multidrug-resistant phenotypes in E. coli belonging to phylogenetic group B2 were significantly higher than those of other groups. This study clarified the distribution of antimicrobial-resistant (AMR) E. coli in Japanese racehorses. A continuous monitoring program for antimicrobial resistance is required to control the spread of AMR bacteria in racehorses.

Antimicrobial-Resistant Enterococcus faecium and Enterococcus faecalis Isolated From Healthy Thoroughbred Racehorses in Japan.

In this study, the occurrence of antimicrobial-resistant (AMR) enterococci was evaluated in Thoroughbred (TB) racehorses in Japan. Fecal samples were collected from 212 healthy TB racehorses at the Miho and Ritto Training Centers of the Japan Racing Association from March 2017 to August 2018. Isolation and identification were performed by enterococcus selective medium and confirmed to the species using MALDI-TOF MS. Enterococcus faecium and E. faecalis isolates were subjected to antimicrobial susceptibility test against 11 antimicrobials by minimum inhibitory concentration based on recommendation from Clinical and Laboratory Standards Institute guidelines. Among 583 enterococcus isolates, E. faecium and E. faecalis were identified for 48.2% (281/583) and 7.4% (43/583), respectively. One isolate that was representing E. faecium (153 isolates) and E. faecalis (31 isolates) from each sample was selected for antimicrobial susceptibility test. The highest rate of resistance for E. faecium isolates was observed against enrofloxacin (57.5%; 88/153), followed by streptomycin (32.0%; 49/153), kanamycin (18.3%; 28/153), gentamycin (5.9%; 9/153), erythromycin (5.9%; 9/153), and oxytetracycline (4.6%; 7/153). For E. faecium isolates, the highest resistance was observed against streptomycin (90.3%; 28/31), followed by kanamycin (41.9%; 13/31), gentamycin (29.0%; 9/31), lincomycin (9.7%; 3/31), oxytetracycline (6.5%; 2/31), erythromycin (6.5%; 2/31), tylosin (6.5%; 2/31), enrofloxacin (6.5%; 2/31), and chloramphenicol (3.2%; 1/31). The results indicated that enrofloxacin and aminoglycosides were highly resistant among tested antimicrobials. Continuous monitoring studies are useful to increase the awareness of the potential for AMR bacteria to arise from imprudent use of antimicrobials in TB racehorses in Japan.

Extended-spectrum ß-lactamase-producing Escherichia coli isolated from healthy Thoroughbred racehorses in Japan.

Extended-spectrum ß-lactamase-producing Escherichia coli (ESBLEC) have become a major health concern in both human and veterinary medicine. These bacteria could become a critical problem in equine medicine due to the limited number of antimicrobial drugs available. However, there are no previous reports of ESBLEC isolated from horses in Japan. The objectives of this study were to investigate the occurrence of ESBLEC isolated from feces in healthy Thoroughbred racehorses in Japan. Feces samples were collected from 147 healthy Thoroughbred racehorses by equine veterinarians at the Japan Racing Association (103 from Miho Training Center and 44 from Ritto Training Center) between March 2017 and April 2018. Samples were screened for ESBLECs using MacConkey agar supplemented with 1 µg/ml cefotaxime. Detection of ESBL genes was performed by PCR and confirmed by DNA sequencing. Horizontal transmission was demonstrated by conjugation assay. In this study, 24 ESBLECs were isolated from twelve horse feces samples (8.2%). All ESBLECs harbored blaCTX-M-2, and both blaTEM-1 and blaCTX-M-2 were detected in nine isolates (37.5%). ESBLECs showed resistance to all ß-lactam antibiotics (100%) tested, followed by trimethoprim (66.7%), streptomycin (62.5%), tetracycline (25.0%), and oxytetracycline (25.0%). Horizontal transmission was successfully demonstrated by conjugation assay in eight of 13 isolates, and blaCTX-M-2 was detected by PCR in all transconjugants. This study showed that racehorses in Japan are potential reservoirs of ESBLECs.

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.

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.

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.

Treatment of recent-onset atrial fibrillation with quinidine and flecainide in Thoroughbred racehorses: 107 cases (1987-2014).

OBJECTIVE To compare the efficacy of quinidine and flecainide in treating naturally occurring, recent-onset atrial fibrillation (AF) in Thoroughbred racehorses. DESIGN Retrospective case series. ANIMALS 107 Thoroughbred racehorses. PROCEDURES Medical records of racehorses with AF that were treated with quinidine or flecainide between 1987 and 2014 were reviewed. Signalment, history, treatments, complications, and outcome data were collected. Horses were allocated to 2 groups according to the initial treatment: initial treatment with quinidine (group 1) or initial treatment with flecainide (group 2). Horses in group 2 that did not convert to sinus rhythm with flecainide were then administered quinidine (group 3). Complications, total quinidine dose, and duration of treatment were compared. Rates of conversion for horses treated with quinidine versus flecainide were also compared. RESULTS Overall rate of cardioversion was 91% (97/107). There was a significant difference in the rate of cardioversion for quinidine alone (91% [71/78]), compared with flecainide alone (41% [12/29]). In group 3, the conversion rate after the addition of quinidine treatment was 82% (14/17). Total quinidine dose and treatment duration did not differ significantly between groups 1 and 3. CONCLUSIONS AND CLINICAL RELEVANCE Overall rate of cardioversion for Thoroughbred racehorses with AF was similar to that in previous reports. Flecainide treatment was less effective than quinidine treatment, but the frequency of complications did not differ between quinidine and flecainide. Further investigation is suggested to evaluate the efficacy of flecainide for cardioversion in athletic horses.

Clinical usefulness of intravenous constant rate infusion of fentanyl and medetomidine under sevoflurane anesthesia in Thoroughbred racehorses undergoing internal fixation surgery.

A total of 20 racehorses with longitudinal fractures underwent internal fixation surgery under sevoflurane anesthesia combined with infusion of medetomidine (3 µg/kg/hr) alone (10 horses, group M) or medetomidine and fentanyl (7 µg/kg/hr) (10 horses, group FM). In group FM, the end-tidal sevoflurane concentration during surgery was maintained significantly lower than in group M (2.8-2.9% for group M vs. 2.2-2.6% for group FM, P<0.01). The mean arterial blood pressure was maintained over 70 mmHg using dobutamine infusion (group M, 0.36-0.54 µg/kg/min; group FM, 0.27-0.65 µg/kg/min), and the recovery qualities were clinically acceptable in both groups. In conclusion, co-administration of fentanyl and medetomidine by constant rate infusion may be a clinically useful intraoperative anesthetic adjunct for horses to reduce the requirement of sevoflurane when they undergo orthopedic surgery.

Effects of pre-shipping enrofloxacin administration on fever and blood properties in adult Thoroughbred racehorses transported a long distance.

To evaluate the effects of single-dose enrofloxacin (ERFX) on fever and blood properties in 68 Thoroughbred racehorses after long-distance transportation, horses were assigned to receive ERFX (5 mg/kg, IV; ERFX group; n=52) or saline (0.9% NaCl) solution (50 ml, IV; control group; n=16) ≤1 hr before transportation. Horses were transported 1,122 km using commercial vans over the course of approximately 21 hr. Clinical examinations and hematologic analyses were performed before and after transportation. Rectal temperatures, white blood cell counts and serum amyloid A concentration of ERFX group were significantly lower than control group (P<0.01, P<0.01 and P<0.05, respectively). In conclusion, these results show ERFX administration just before transportation is effective at preventing transportation-associated fever in adult Thoroughbred racehorses.

Effects of pre-shipping marbofloxacin administration on fever and blood properties in healthy Thoroughbreds transported a long distance.

The present study evaluated the effects of single-dose marbofloxacin in protecting horses against fever associated with transportation using 48 healthy Thoroughbreds. All horses were premedicated with interferon-α (0.5 U/kg, sublingually, every 24 hr) for 2 days before transportation and on the day of transportation. Horses were randomly assigned to receive marbofloxacin (2 mg/kg, IV, once; MRFX group), enrofloxacin (5 mg/kg, IV, once; ERFX group) or saline (0.9% NaCl) solution (10 ml, IV, once; control group) ≤1 hr before being transportation. Each group contained 16 horses (8 males, 8 females). Horses were transported 1,210 km using commercial vans over the course of approximately 26 hr. Clinical examinations and hematologic analyses were performed on all horses both before and after transportation. Post-transportation neutrophil to lymphocyte ratios were significantly lower in horses in the MRFX group compared with the control horses. The serum amyloid A levels were significantly lower in horses in the MRFX group and ERFX group compared with the control horses. Regarding the post-transportation rectal temperatures, fever was detected in 0 horses and 1 horse in the MRFX and ERFX groups, respectively, whereas fevers exceeding 39.1°C were detected in 2 horses in the control group. Additionally, the number of essential post-transportation treatments provided by veterinarians was reduced 3-fold in the MRFX and ERFX groups compared with the saline group. MRFX provided ERFX-like protection against fever associated with long-distance transportation, yielding significantly better protection than saline. Administration of MRFX just before transportation deserves a further study for efficacy in preventing horse fever associated with transportation.

Cardiovascular effects of dobutamine and phenylephrine infusion in sevoflurane-anesthetized Thoroughbred horses.

To determine dose-dependent cardiovascular effects of dobutamine and phenylephrine during anesthesia in horses, increasing doses of dobutamine and phenylephrine were infused to 6 healthy Thoroughbred horses. Anesthesia was induced with xylazine, guaifenesin and thiopental and maintained with sevoflurane at 2.8% of end-tidal concentration in all horses. The horses were positioned in right lateral recumbency and infused 3 increasing doses of dobutamine (0.5, 1.0 and 2.0 µg/kg/min) for 15 min each dose. Following to 30 min of reversal period, 3 increasing doses of phenylephrine (0.25, 0.5 and 1.0 µg/kg/min) were infused. Cardiovascular parameters were measured before and at the end of each 15-min infusion period for each drug. Blood samples were collected every 5 min during phenylephrine infusion period. There were no significant changes in heart rate throughout the infusion period. Both dobutamine and phenylephrine reversed sevoflurane-induced hypotension. Dobutamine increased both mean arterial blood pressure (MAP) and cardiac output (CO) as the result of the increase in stroke volume, whereas phenylephrine increased MAP but decreased CO as the result of the increase in systemic vascular resistance. Plasma phenylephrine concentration increased dose-dependently, and these values at 15, 30 and 45 min were 6.2 ± 1.2, 17.0 ± 4.8 and 37.9 ± 7.3 ng/ml, respectively.

Hemodynamic effects of 6% hydroxyethyl starch infusion in sevoflurane-anesthetized thoroughbred horses.

To determine hemodynamic effects of hydroxyethyl starch (HES) infusion during anesthesia in horses, incremental doses of 6% HES were administered to 6 healthy Thoroughbred horses. Anesthesia was induced with xylazine, guaifenesin and thiopental and maintained with sevoflurane at 2.8% of end-tidal concentration in all horses. The horses were positioned in right lateral recumbency and administered 3 intravenous dose of 6% HES (5 ml/kg) over 15 min with 15-min intervals in addition to constant infusion of lactated Ringer's solution at 10 ml/kg/hr. Hemodynamic parameters were measured before and every 15 min until 90 min after the administration of 6% HES. There was no significant change in heart rate and arterial blood pressures throughout the experiment. The HES administration produced significant increases in mean right atrial pressure, stroke volume, cardiac output (CO) and decrease in systemic vascular resistance (SVR) in a dose-dependent manner. There was no significant change in electrolytes (Na(+), K(+), Cl(-)) throughout the experiment, however, packed cell volume, hemoglobin concentration, and total protein and albumin concentrations decreased in a dose-dependent manner following the HES administration. In conclusion, the HES administration provides a dose-dependent increase in CO, but has no impact upon arterial blood pressures due to a simultaneous decrease in SVR.

Characteristic of inflammatory airway disease in Japanese thoroughbred racehorses.

Inflammatory airway disease (IAD) is a common cause of poor performance, interruption of training and premature retirement in racehorses. It is also reported that up to 80% of horses are affected at some point in the first years of training in UK and Australia. However, no studies with regard to the information on occurrence of IAD in Japanese Thoroughbred racehorses have been reported. To investigate the occurrence and the characteristics of IAD, epidemic research including endoscopic examination of the airway tract and trachea wash was conducted for Thoroughbred racehorses presenting coughs or poor performance which airway tract disease was suspected stalled in training facility managed by Japan Racing Association. Fifty-six out of 76 Thoroughbred racehorses (73.7%) presenting coughing or poor performance were diagnosed as IAD. Mean incidence rate of IAD was 0.3% and it has been confirmed that constant number of IAD exists in Japan. Up to 35.7% of IAD horses showed upper airway abnormalities in some extent. There was a trend for IAD horses to use wood shavings for bedding and fed hay from the ground compared with the control group. Therefore, improvement of stabling environment may aid in preventing IAD. This study demonstrated that Japanese Thoroughbred racehorses are affected by IAD likewise other countries as well as demonstrated the characteristics of IAD which may contribute to the clarification of the pathogenesis of IAD.

Tracheal Endoscopic and Cytological Findings and Blood Examination Results in Thoroughbred Racehorses Suspected to have Lower Respiratory Tract Disease.

Cytology of tracheal aspirates, tracheal endoscopic and blood tests were carried out to 86 Thoroughbred racehorses presenting coughs or poor performance which were suspected to have lower respiratory tract disease (LRTD) to assess the conditions of the disorders. Racehorses were classified into coughing (66 horses) and non-coughing (20 horses) groups based on clinical symptoms. Nine Thoroughbred racehorses without respiratory abnormality were used as controls. Assessment of grades of airway mucus, cytology of tracheal aspirates and serum amyloid A (SAA), fibrinogen (Fbg) and pulmonary surfactant protein D (SP-D) measurements were performed. Relationships between age, gender and racing careers were also investigated to understand the characteristics of LRTD in racehorses. Mean age was significantly higher in non-coughing group compared to coughing group. Existence of racing career and number of starts were significantly greater in non-coughing group compared to coughing group. On the other hand, grades of airway mucus were significantly higher in coughing group compared to control group. Percentages of neutrophils in tracheal aspirates were significantly higher in coughing group compared to non-coughing and control groups. SAA, Fbg and SP-D were higher in coughing group compared to non-coughing and control groups indicating that condition of coughing group is in the acute phase. Positive rate of inflammatory airway disease was significantly higher in coughing and non-coughing groups compared to control group. It was concluded that carrying out comprehensive evaluation including investigation on SAA, Fbg and SP-D analysis with airway assessment to Thoroughbred racehorses which were suspected to have LRTD are useful procedure to understand the pathological condition which aid to initiate appropriate treatment, prognosis judgment or to advise trainers to consider altering training regimen.