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.

Multiplex Detection of Transgenes Using πCode Technology for Gene Doping Control.

To ensure fair competition and sports integrity, gene doping is prohibited in horseracing and equine sports. One gene doping method is by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable for multiplex detection. In this proof-of-concept study, we developed a highly sensitive and multiplex transgene detection method using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) multiplex polymerase chain reaction amplification of 12 targeted transgenes in a single tube, (2) detection using a mixture of 12 probes labeled with different πCodes, and (3) median fluorescence intensity measurement of fluorescent πCodes. Twelve transgenes cloned into plasmid vectors were targeted, and 1500 copies of each plasmid were spiked into 1.5 mL of horse plasma. Subsequently, a novel method using πCode succeeded in detecting all the transgenes using their DNA extracts. Additionally, we detected the erythropoietin (EPO) transgene in blood samples from a horse administered solely with the EPO transgene using this method. Therefore, the πCode detection method is considered suitable for multitarget gene detection in gene doping tests.

Identification of Potential miRNA Biomarkers to Detect Hydrocortisone Administration in Horses.

Circulating microRNAs (miRNAs) are stable in bodily fluids and are potential biomarkers of various diseases and physiological states. Although several studies have been conducted on humans to detect drug doping by miRNAs, research on drugs and miRNAs in horses is limited. In this study, circulating miRNAs in horses after hydrocortisone administration were profiled and variations in miRNAs affected by hydrocortisone administration during endogenous hydrocortisone elevation were examined. The miRNAs were extracted from thoroughbred horse plasma before and after hydrocortisone administration and subjected to small RNA sequencing and reverse transcription quantitative PCR (RT-qPCR). RT-qPCR validation was performed for the 20 miRNAs that were most affected by hydrocortisone administration. The effects of elevated endogenous hydrocortisone levels due to exercise and adrenocorticotropic hormone administration were also confirmed. The validation results showed that approximately half of the miRNAs showed the same significant differences as those obtained using small RNA sequencing. Among the twenty miRNAs, two novel miRNAs and miR-133a were found to vary differently between exogenous hydrocortisone administration and endogenous hydrocortisone elevation. This study provides basic knowledge regarding the circulating miRNA profile of horses after hydrocortisone administration and identifies three miRNAs that could potentially be used as biomarkers to detect hydrocortisone administration.

Monitoring the positive conversion of anti-erythrocyte antibodies in blood transfusion donor horses.

To confirm the positive conversion of antibodies against erythrocyte antigens in horses, possible blood transfusion donor horses selected from draft horse populations were periodically monitored with an indirect antiglobulin (Coombs) test for approximately 3 years. In this study, 19 horses (16 females and 3 males) were investigated, and five mares showed alloantibodies during the monitoring period. Four mares were typically pregnant when positive conversion was detected, whereas no particular cause of conversion could be observed for one mare based on its clinical records. In the analyzed horses, most positive conversions were possibly due to pregnancy, as conversion occurred more often during this period than after parturition. Pregnancy is considered a key event for positive conversion. Additionally, in cases in which unknown causative sensitization is confirmed, continuous monitoring with a test to detect antibodies should be performed, even if the possible donor is selected and maintained.

Construction of an individual identification panel for horses using insertion and deletion markers.

Individual identification and paternity testing are important for avoiding inbreeding in the management of small populations of wild and domestic animals. In horse racing industries, they are extremely important for identifying and registering individuals and doping control to ensure fair competition. In this study, we constructed an individual identification panel for horses by using insertion and deletion (INDEL) markers. The panel included 39 INDEL markers selected from a whole-genome INDEL database. Genotyping of 89 Thoroughbreds showed polymorphisms with minor allele frequencies (MAFs) of 0.180-0.489 in all markers. The total probability of exclusion for paternity testing, power of discrimination, and probability of identity were 0.9994271269, >0.9999999999, and 0.9999999987, respectively. The panel was applied to 13 trios (sires, dams, and foals), and no contradictions were observed in genetic inheritance among the trios. When this panel was applied to the trios (52 trios) containing false fathers, an average of 7.3 markers excluded parentage relationships. In addition, genomic DNA extracted from the urine of six horses was partially genotyped for 39 markers, and 6-28 markers were successfully genotyped. The newly constructed panel has two advantages: a low marker mutation rate compared with short tandem repeats and a genotyping procedure that is as simple as short tandem repeat typing compared with single nucleotide variant typing. This panel can be applied for individual identification, paternity determination, and urine-sample identification in Thoroughbred horses.

Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests.

Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, sufficient qPCR detection was demonstrated after refrigeration for 1 to 2 weeks post collection. For sample B, the following procedures were confirmed to be suitable for storage: 1) centrifugation after sample receipt, 2) frozen storage, 3) natural thawing at room temperature, and 4) centrifugation without mixing blood cell components. Our results indicated that long-term cryopreservation yielded good plasma components from frozen blood samples even though it destroyed blood cells, indicating its applicability to the gene doping test using sample B, which can be stored for later use. Sample storage procedures are as important as detection methods in doping tests. Therefore, the series of procedures that we evaluated in this study will contribute to the efficient performance of gene doping tests through qPCR using blood samples.

Additional studies on nicotine exposure in horses: Accurate quantification and elimination profiles of potential biomarkers in plasma and urine.

RATIONALE: For the purpose of doping control, this is the first report of accurate quantification of four critical structural isomers of nicotine metabolites (trans-3'-hydroxycotinine, cis-3'-hydroxycotinine, 5'-hydroxycotinine, and N'-hydroxymethylnorcotinine) in equine plasma and urine for the establishment of their elimination profiles. Besides, the pharmacokinetic studies of trans-3'-hydroxycotinine and N'-hydroxymethylnorcotinine in equine plasma and urine are also presented for the first time. METHODS: The accurate quantification methods of the aforementioned four structural isomers in horse plasma and urine were successfully developed and validated using the solid-phase extractions followed by liquid chromatography/tandem mass spectrometry analysis. Baseline chromatographic separation was achieved to completely differentiate these isomers, which shared the same selected reaction monitoring transition. Such methods were applied to post-administration samples obtained from the nicotine and tobacco leaf administration studies for the establishment of pharmacokinetic profiles. RESULTS: N'-Hydroxymethylnorcotinine could be quantified for the longest period, ranging from 48 to 72 h in plasma and 96 h in urine after a single administration of 250 mg of nicotine and an equivalent amount of nicotine in tobacco leaves. In terms of detection, both N'-hydroxymethylnorcotinine and trans-3'-hydroxycotinine could be detected up to the last sample collection time point (96 h), indicating that they are the most appropriate biomarkers for nicotine exposure. CONCLUSIONS: N'-Hydroxymethylnorcotinine and trans-3'-hydroxycotinine were detected longest in plasma and urine samples after both nicotine and tobacco leaf administrations, and N'-hydroxymethylnorcotinine was deemed most appropriate as a monitoring target due to its relatively higher abundance and slower elimination rate. These two biomarkers could also be used to differentiate sample contamination by tobacco products and genuine nicotine exposure to horse regardless of intentionality.

Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene-doping detection considering the presence of pseudogenes.

Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome. In this study, we identified 62 processed pseudogene candidates as intron-less genes from whole-genome sequencing (WGS) data of Thoroughbred horses using delly structural variation software. The 62 processed pseudogene candidates were confirmed by PCR amplification of intron-less products. A total of 11 processed pseudogenes were confirmed in the genome of all 23 analysed horses, whereas three processed pseudogenes with structures of ATP11B, DPH3 and RPL17 were detected in only one of 115 horses by PCR amplification of intron-less products. Currently, most of the gene doping tests proposed in human and horse sports are adapted PCR-based methods using hydrolysis probes to detect exon/exon junctions in transgenes because the operation is simple and economical. However, when the pseudogene is present in the host genome, the PCR-based methods may have a potential risk of detecting false positives. In this study, because processed pseudogenes that exist less frequently in the horse genome may affect PCR-based transgene detection in gene-doping tests, we propose and demonstrate that PCR amplification and sequencing using primers designed on transgene and promotors and/or polyadenylation signal for gene expression are useful for gene-doping detection as an additional confirmatory test to prevent false positives.

Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control.

Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetically modified horse. Genome-wide screening of non-targeted transgenes was performed to find structural variation using DELLY based on split-read and paired-end algorithms and Control-FREEC based on read-depth algorithm. We detected the EPO transgene as an intron deletion in the WGR data by the split-read algorithm of DELLY. In addition, single-nucleotide polymorphisms and insertions/deletions artificially introduced in the EPO transgene were identified by WGR. Therefore, genome-wide screening using WGR can contribute to gene-doping control even if the targets are unknown. This is the first study to detect transgenes as intron deletions for gene-doping detection.

Robustness of Digital PCR and Real-Time PCR in Transgene Detection for Gene-Doping Control.

Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time PCR assays. Digital PCR yielded a similar copy number regardless of the probe (P-true: 1230.7, P-false: 1229.7), whereas real-time PCR revealed a decrease in sensitivity based on Cq values (P-true: 23.5, P-false: 29.7). When substituted primers were used, the detected copy number decreased in the digital PCR assay, and the Cq value in real-time PCR was much higher. Interestingly, digital PCR copy numbers improved by performing PCR at a low annealing temperature, even if a substituted probe was used. Thus, when primer and probe sequences did not completely match the template transgene, digital PCR was relatively robust, but real-time PCR was less sensitive. Although PCR specificity may be reduced, PCR sensitivity can be improved by lowering the annealing temperature. If the target sequence is substituted to escape doping detection, it may be desirable to set the annealing temperature lower and use a more robust method, such as digital PCR, to increase the detection of positive cases, which will also result in fewer false-negative results.

Investigation of erythrocyte antigen frequencies in draft horse populations in Japan to assess blood donor suitability.

Erythrocyte alloantigen frequencies of draft horses in Japan were investigated to assess blood donor suitability for transfusion. Here, 148 Japanese draft, 69 Percheron, and 65 Breton horses were blood-typed and subjected to an indirect antiglobulin test. Regarding the major immunogenic factors, the rates of Aa- and Qa-negative horses ranged from 0.35 to 0.49 and from 0.82 to 1.00, respectively. The rate of alloantibody-positive horses ranged from 0.12 to 0.35. Although the prevalence of alloantibodies in these horses was higher than that expected naturally, the rates of Aa- and Qa-negative horses were higher than those of some breeds reported previously. The current draft horse population could provide potential candidates for donors, and the obtained information may contribute to the selection of a safe donor for transfusion.

Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests.

One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR.

Utility of systemic voriconazole in equine keratomycosis based on pharmacokinetic-pharmacodynamic analysis of tear fluid following oral administration.

OBJECTIVE: To clarify the detailed pharmacokinetics (PK) of orally administered voriconazole in tear fluid (TF) of horses for evaluating the efficacy of voriconazole secreted into TF against equine keratomycosis. ANIMALS STUDIED: Five healthy Thoroughbred horses. PROCEDURES: Voriconazole was administrated through a nasogastric tube to each horse at a single dose of 4.0 mg/kg. TF and blood samples were collected before and periodically throughout the 24 hours after administration. Voriconazole concentrations in plasma and TF samples were analyzed using liquid chromatography-electrospray tandem-mass spectrometry. The predicted voriconazole concentration in both samples following multiple dosing every 24 hours was simulated by the superposition principle. RESULTS: The mean maximum voriconazole concentrations in plasma and TF were 3.3 µg/mL at 1.5 h and 1.9 µg/mL at 1.6 h, respectively. Mean half-life in both samples were 16.4 and 25.2 h, respectively. The ratio of predicted AUC0-24 at steady state in TF (51.3 µg∙h/mL) to previously published minimum inhibitory concentration (MIC) of Aspergillus and Fusarium species was >100 and 25.7, respectively. CONCLUSIONS: This study demonstrated the detailed single-dose PK of voriconazole in TF after oral administration and simulated the predicted concentration curves in a multiple oral dosing. Based on the analyses of PK-PD, the simulation results indicated that repeated oral administration of voriconazole at 4.0 mg/kg/d achieves the ratio of AUC to MIC associated with treatment efficacy against Aspergillus species. The detailed PK-PD analyses against pathogenic fungi in TF can be used to provide evidence-based medicine for equine keratomycosis.

Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports.

Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded, the quality of genomic DNA from samples stored for years at refrigerated temperatures (4-8°C) was maintained. High-molecular-weight genomic DNA was isolated from hair roots using a magnetic silica beads method of extraction, enabling WGR from horsehair root extracts. Nucleotide sequencing results and numbers of single-nucleotide polymorphisms and insertions/deletions concurred with those previously reported for WGR of DNA extracted from whole blood. Therefore, we consider that storing hair samples at refrigerated temperatures prevents degradation of DNA, allowing the detection of gene doping in these samples based on WGR. It is likely this finding will also have a deterrent effect, as it is now possible to test horses with archived samples even if they or their parents are deceased. To our knowledge, this is the first report employing WGR on horsehair roots stored for a long term.

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.

Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions.

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs.

Distribution of Y chromosomal haplotypes in Japanese native horse populations.

The distribution of Y chromosomal haplotypes in Japanese native horse populations was investigated to obtain genetic information on these populations. Here, 159 male/gelded horses from eight local populations were investigated, and three Y haplotypes (JHT-1, JHT-2, and JHT-3) were identified by analyzing five Y-linked loci. Five populations had only JHT-1, whereas two populations had only JHT-2. One population had JHT-1 and JHT-3. Based on the geographical distribution of these haplotypes and previously reported haplotypes for other Asian horses, JHT-1 is considered to be a major haplotype in ancestral native horses. The fixation of each haplotype suggests the influence of independent breeding and genetic drift in each population. These findings complement the results from previous genetic studies of Japanese native horses.

A genome-wide association study for body weight in Japanese Thoroughbred racehorses clarifies candidate regions on chromosomes 3, 9, 15, and 18.

Body weight is an important trait to confirm growth and development in humans and animals. In Thoroughbred racehorses, it is measured in the postnatal, training, and racing periods to evaluate growth and training degrees. The body weight of mature Thoroughbred racehorses generally ranges from 400 to 600 kg, and this broad range is likely influenced by environmental and genetic factors. Therefore, a genome-wide association study (GWAS) using the Equine SNP70 BeadChip was performed to identify the genomic regions associated with body weight in Japanese Thoroughbred racehorses using 851 individuals. The average body weight of these horses was 473.9 kg (standard deviation: 28.0) at the age of 3, and GWAS identified statistically significant SNPs on chromosomes 3 (BIEC2_808466, P=2.32E-14), 9 (BIEC2_1105503, P=1.03E-7), 15 (BIEC2_322669, P=9.50E-6), and 18 (BIEC2_417274, P=1.44E-14), which were associated with body weight as a quantitative trait. The genomic regions on chromosomes 3, 9, 15, and 18 included ligand-dependent nuclear receptor compressor-like protein (LCORL), zinc finger and AT hook domain containing (ZFAT), tribbles pseudokinase 2 (TRIB2), and myostatin (MSTN), respectively, as candidate genes. LCORL and ZFAT are associated with withers height in horses, whereas MSTN affects muscle mass. Thus, the genomic regions identified in this study seem to affect the body weight of Thoroughbred racehorses. Although this information is useful for breeding and growth management of the horses, the production of genetically modified animals and gene doping (abuse/misuse of gene therapy) should be prohibited to maintain horse racing integrity.

Sequence variants of BIEC2-808543 near LCORL are associated with body composition in Thoroughbreds under training.

Ligand-dependent nuclear receptor compressor-like (LCORL) encodes a transcription factor, and its polymorphisms are associated with measures of skeletal frame size and adult height in several species. Recently, the single nucleotide polymorphism (SNP) BIEC2-808543 located upstream of LCORL was identified as a genetic diagnostic marker associated with withers height in Thoroughbreds. In this study, 322 Thoroughbreds-in-training were genotyped for BIEC2-808543 to evaluate the association between genotype and body composition traits, including body weight, withers height, the ratio of body weight to withers height, chest circumference, and cannon circumference. Of these, withers height and cannon circumference were significantly associated with LCORL genotypes throughout almost the entire training period in males and females. Animals with a C/T genotype had higher withers height (maximum differences of 1.8 cm and 2.1 cm in males and females, respectively) and cannon circumstance (maximum differences of 0.65 cm and 0.48 cm in males and females, respectively) compared with animals with a T/T genotype. These results suggested that the regulation of LCORL expression influences the skeletal frame size in Thoroughbreds and thus, indirectly affects the body weight. Although LCORL and BIEC2-808543 would be useful for selective breeding in Thoroughbreds, the production of genetically modified animals and gene doping based on genetic information should be prohibited in order to maintain racing integrity.