Rationale
The use of GW1516, a peroxisome proliferator‐activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about ...its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine.
Methods
Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein‐precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high‐resolution mass spectrometry (LC/ESI‐HRMS) with a Q‐Exactive mass spectrometer. Monoisotopic ions of GW1516 and its metabolites were monitored from the full‐scan mass spectral data of pre‐ and post‐administration samples. A quantification method was developed and validated to establish the excretion profiles of GW1516, its sulfoxide, and its sulfone in equine urine.
Results
GW1516 and its nine metabolites including GW1516 sulfoxide, GW1516 sulfone, 5‐(hydroxymethyl)‐4‐methyl‐2‐(4‐trifluoromethylphenyl)thiazole (HMTT), methyl 4‐methyl‐2‐4‐(trifluoromethyl)phenyl‐1,3‐thiazole‐5‐carboxylate (MMTC), 4‐methyl‐2‐4‐(trifluoromethyl)phenyl‐1,3‐thiazole‐5‐carboxylic acid (MTTC), and M1 to M4 were detected in post‐administration urine samples. GW1516 sulfoxide and GW1516 sulfone showed the longest detection times in post‐administration urine samples and were therefore recommended as potential screening targets for doping control purposes. Quantitative analysis was also conducted to establish the excretion profiles of GW1516 sulfoxide and GW1516 sulfone in urine.
Conclusions
For the purposes of doping control of GW1516, the GW1516 sulfoxide and GW1516 sulfone metabolites are recommended as the target analytes to be monitored in equine urine due to their high specificities, long detection times (1 and 4 weeks, respectively), and the ready availability of their reference materials.
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.
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.
Patients with chronic obstructive pulmonary disease (COPD) who are hospitalized are more likely to die from their illness and have increased likelihood of re-admission than those who are not. ...Subsequent re-admissions further increase the burden on healthcare systems. This study compared inpatient admission rates and time-to-first COPD-related inpatient admission among Medicare beneficiaries with COPD indexed on umeclidinium/vilanterol (UMEC/VI) versus tiotropium (TIO).
This retrospective study used the All-Payer Claims Database to investigate hospital admission and re-admission outcomes in Medicare beneficiaries with COPD with an initial pharmacy claim for UMEC/VI or TIO from 1 January 2015 to 28 February 2020. Inpatient admissions, baseline, and follow-up variables were assessed in patients indexed on UMEC/VI and TIO after propensity score matching (PSM), with time-to-first on-treatment COPD-related inpatient admission as the primary endpoint. Re-admissions were assessed among patients with a COPD-related inpatient admission in the 30- and 90-days post-discharge.
Post-PSM, 7152 patients indexed on UMEC/VI and 7069 on TIO were eligible for admissions analysis. The mean (standard deviation SD) time-to-first COPD-related inpatient admission was 46.71 (87.99) days for patients indexed on UMEC/VI and 44.96 (85.90) days for those on TIO (p=0.06). The mean (SD) number of inpatient admissions per patient was 1.24 (2.92) for patients indexed on UMEC/VI and 1.26 (3.05) for those on TIO (p=0.49). Proportion of patients undergoing re-admissions was similar between treatments over both 30 and 90 days, excluding a significantly lower proportion of patients indexed on UMEC/VI than those indexed on TIO for COPD-related re-admissions for hospital stays of 4-7 days and 7-14 days, and all-cause re-admissions for stays of 4-7 days.
Patients with COPD using Medicare in the US and receiving UMEC/VI or TIO reported similar time-to-first inpatient admission and similar proportion of re-admissions.
In the context of doping control, conventional direct chemical testing detects only a limited scope of target substances in equine biological samples. To expand the ability to detect doping agents ...and their detection windows, metabolomics has recently become a common approach for monitoring alteration of biomarkers caused by doping agents in relevant metabolic pathways. In horse racing, remarkable changes in metabolic profiles between the rest state and racing are likely to affect the identification of doping biomarkers. Previously, we reported a limited number of significantly upregulated metabolites after racing, based on a non‐targeted metabolomics approach using out‐of‐competition and post‐race equine plasma samples. In this study, we performed a more thorough analysis of the data set, using pathway analysis to establish a post‐race biomarkers database (PBD) that includes upregulated and downregulated metabolites, their fold changes, and relevant pathways, with the main objective of improving our understanding of changes in physiological status related to horse racing. Statistical analysis of the PBD revealed that two peak combinations of pentadecanoyl carnitine/galactosylglycerol (P/G) and heptadecanoyl carnitine/galactosylglycerol (H/G) could be used as potential biomarkers for the discrimination of the rest and post‐race groups. To demonstrate the applicability of the PBD, we validated the post‐race biomarkers P/G and H/G (highly involved in lipid metabolism) by a single‐blind test. This strategy, which combines establishment of a biomarker database with pathway analysis, represents a powerful tool for discovering potential doping biomarkers in the future.
Metabolomics has become a common approach for the purpose of doping control in the horse racing. Metabolic changes between the rest state and racing are very significant and are likely to affect the identification of potential doping biomarkers. In this study, a post‐race biomarkers database (PBD) was established, and potential post‐race biomarkers were successfully identified.
Rationale To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of ...target substances at the required detection levels in equine urine. Methods The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid‐phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed‐phase column and analyzed using liquid chromatography/high‐resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run. Results The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples. Conclusions We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub‐ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full‐scan mode.
Rationale
GW1516 is a peroxisome proliferator‐activated receptor‐δ (PPAR‐δ) agonist that is banned in horseracing and equestrian sports. Long‐term detection and longitudinal distribution of GW1516 in ...the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control.
Methods
Mane hairs were divided into three segments (0–7, 7–15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were analyzed by liquid chromatography/electrospray ionization high‐resolution mass spectrometry (LC/ESI‐HRMS) using a Q‐Exactive instrument. This method was successfully validated and applied to post‐administration samples to confirm the presence of GW1516 and its metabolites and estimate the uptake amounts of GW1516.
Results
After administration of 150 mg of GW1516 to a thoroughbred mare, GW1516 was detected in one of two segments of all mane hairs, and four metabolites, namely GW1516 sulfoxide, GW1516 sulfone, 5‐(hydroxymethyl)‐4‐methyl‐2‐(4‐trifluoromethylphenyl)thiazole (HMTT), and 4‐methyl‐2‐4‐(trifluoromethyl)phenyl‐1,3‐thiazole‐5‐carboxylic acid (MTTC), were also identified. The longitudinal distribution analysis results showed that the maximum uptake of GW1516 into hair (approximately 0.05 pg/mg) was observed at around 13 weeks post‐administration and GW1516 could be detected and confirmed up to 6 months post‐administration.
Conclusions
The parent drug GW1516 was identified as the most appropriate monitoring target in equine hair for controlling its misuse in horses. The use of hair analysis could extend the detection time of GW1516 to at least 6 months after the administration of 150 mg of GW1516 to a thoroughbred mare.
Indoor positioning system (IPS) enables real-time tracking and positioning within indoor environments and supports various location-based services (LBSs) across different application settings. The ...growth of the Internet of Things (IoT) has enabled fingerprint-based IPS to achieve sub-meter or centimeter-level accuracy. However, packet loss issues caused by attenuation, interference from other devices, and noise hinder the fingerprint-based IPS. Despite developments in IPS methods, such as fingerprint augmentation and the integration of variational inference, effectively using these techniques with fingerprints under packet loss to achieve robust positioning continues to pose a challenge. This article develops a real-time positioning model named Packet Loss Indoor Positioning Net (PLIPNet). PLIPNet combines the variational inference process and encodes statistical means of fingerprints in each location as prior distributions of latent variables, making it free from prior parameter configurations. The location's spatial information is incorporated into latent space and probability representation to improve latent distribution distinguishability and avoid significant positioning errors. Comparisons with state-of-the-art positioning models show that PLIPNet consistently performs the best under various packet loss settings. For instance, when the packet loss rate reaches 80%, PLIPNet achieves a localization error of only 25.7% of that achieved by the best existing model.
We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in MS pathology. In this study, we found that the acrolein scavenger hydralazine (HZ), when applied from the day ...of induction, can suppress acrolein and alleviate motor and sensory deficits in a mouse experimental autoimmune encephalomyelitis (EAE) model. Furthermore, we also demonstrated that HZ can alleviate motor deficits when applied after the emergence of MS symptoms, making potential anti-acrolein treatment a more clinically relevant strategy. In addition, HZ can reduce both acrolein and MPO, suggesting a connection between acrolein and inflammation. We also found that in addition to HZ, phenelzine (PZ), a structurally distinct acrolein scavenger, can mitigate motor deficits in EAE when applied from the day of induction. This suggests that the likely chief factor of neuroprotection offered by these two structurally distinct acrolein scavengers in EAE is their common feature of acrolein neutralization. Finally, up-and-down regulation of the function of aldehyde dehydrogenase 2 (ALDH2) in EAE mice using either a pharmacological or genetic strategy led to correspondent motor and sensory changes. This data indicates a potential key role of ALDH2 in influencing acrolein levels, oxidative stress, inflammation, and behavior in EAE. These findings further consolidate the critical role of aldehydes in the pathology of EAE and its mechanisms of regulation. This is expected to reinforce and expand the possible therapeutic targets of anti-aldehyde treatment to achieve neuroprotection through both endogenous and exogenous manners.
In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity ...purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and
N
-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50 pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of
N
-acetylated LKKTETQ and its metabolite
N
-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10 mg
N
-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses.
Figure
Product-ion scans and mass spectral assignments of the fragment ions of the seven target peptides
