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Calcium dobesilate (CD) is a synthetic venoactive drug used in veterinary medicine to treat equine navicular disease. Etamsylate is a haemostatic agent used in horses for the treatment of exercise-induced pulmonary haemorrhage. Both etamsylate and CD dissociate in the circulatory system with 2,5-HBSA as the active drug. The aim of the research was to be able to provide detection time (DT) advice from pharmacokinetic (PK) studies in Thoroughbred horses to better inform trainers, and their veterinary surgeons, prescribing these substances for treatment of Thoroughbred racehorses. Two (pilot study) and six (final study) horses were given 28 and 9 repeated dose of CD (3 mg/kg BID) respectively. Two horses were each given a single intravenous (IV) dose of etamsylate (10 mg/kg). Plasma and urine 2,5-HBSA concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CD pilot study revealed that steady state could be reached with a few days and that 2,5-HBSA in plasma and urine shows instability during storage at -20°C but appears stable at -80°C. A novel holistic non-linear mixed-effects three-compartmental PK model was developed that described both plasma and urine concentrations of 2,5-HBSA, from either CD or etamsylate administration. Typical values for 2,5-HBSA clearance and bioavailability were 2.0 mL/min/kg and 28% respectively. Using the parameters obtained from this PK model, in conjunction with methodology developed by Toutain, afforded a possible screening limit (SL) that can regulate for a DT of 3 days in urine; however, a corresponding SL in plasma would be below current levels of detection. However, it is the responsibility of the individual racing authorities to apply their own risk management with regard to SLs and DTs., (© 2023 John Wiley & Sons Ltd.)

Samples of the 'dietary supplement' Furazadrol sourced through the internet have been reported to contain the designer anabolic androgenic steroids [1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol (furazadrol F) and [1',2']isoxazolo[4',3':2,3]-5α-androstan-17β-ol (isofurazadrol IF). These steroids contain an isoxazole fused to the A-ring and were designed to offer anabolic activity while evading detection, raising concerns over the potential for abuse of this preparation in sports. The metabolism of Furazadrol (F:IF, 10:1) was studied by in vivo methods in greyhounds. Urinary phase II Furazadrol metabolites were detected as glucuronides after a controlled administration. These phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli β-glucuronidase to afford the corresponding phase I metabolites. Using a library of synthetically derived reference materials, the identities of seven urinary Furazadrol metabolites were confirmed. Major confirmed metabolites were isofurazadrol IF, 4α-hydroxyfurazadrol 4α-HF and 16α-hydroxy oxidised furazadrol 16α-HOF, whereas the minor confirmed metabolites were furazadrol F, 4β-hydroxyfurazadrol 4β-HF, 16β-hydroxyfurazadrol 16β-HF and 16β-hydroxy oxidised furazadrol 16β-HOF. One major hydroxyfurazadrol and two dihydroxyfurazadrol metabolites remained unidentified. Qualitative excretion profiles, limits of detection and extraction recoveries were established for furazadrol F and major confirmed metabolites. These investigations identify the key urinary metabolites of Furazadrol following oral administration, which can be incorporated into routine screening by anti-doping laboratories to aid the regulation of greyhound racing., (© 2021 John Wiley & Sons, Ltd.)

Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17β-ol), reduced and dihydroxylated madol (17α-methyl-5α-androstane-2β,3α,17β-triol), and the isomeric enone metabolites 17β-hydroxy-17α-methyl-5α-androst-3-en-2-one and 17β-hydroxy-17α-methyl-5α-androst-2-en-4-one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell-based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC-MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports., (© 2020 John Wiley & Sons, Ltd.)

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) was investigated, with ATP and Na 2 SO 4 providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17β-hydroxy-2α,17α-dimethyl-5α-androstan-3-one) was performed as a prediction of the in vivo metabolic profile., (Copyright © 2017 John Wiley & Sons, Ltd.)

In recent years, the potential for anabolic steroid abuse in equine sports has increased due to the growing availability of designer steroids. These compounds are readily accessible online in 'dietary' or 'nutritional' supplements and contain steroidal compounds which have never been tested or approved as veterinary agents. They typically have unusual structures or substitution and as a result may pass undetected through current anti-doping screening protocols, making them a significant concern for the integrity of the industry. Despite considerable focus in human sports, until recently there has been limited investigation into these compounds in equine systems. To effectively respond to the threat of designer steroids, a detailed understanding of their metabolism is needed to identify markers and metabolites arising from their misuse. A summary of the literature detailing the metabolism of these compounds in equine systems is presented with an aim to identify metabolites suitable for incorporation into screening protocols by anti-doping laboratories. The future of equine anti-doping research is likely to be guided by the incorporation of alternate testing matrices into routine screening, the improvement of in vitro technologies that can mimic in vivo equine metabolism, and the improvement of instrumentation or analytical methods that allow for the development of untargeted screening, and metabolomics approaches for use in anti-doping screening protocols. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The untargeted detection of phase II metabolites is a key issue for the study of drug metabolism in biological systems. Sensitive and selective mass spectrometric (MS) techniques coupled to ultrahigh performance liquid chromatographic (UHPLC) systems are the most effective for this purpose. In this study, we evaluate different MS approaches with a triple quadrupole instrument for the untargeted detection of bis-sulfate metabolites. Bis-sulfates of 23 steroid metabolites were synthesized and their MS behavior was comprehensively studied. Bis-sulfates ionized preferentially as the dianion ([M - 2H] 2- ) with a small contribution of the monoanion ([M - H] - ). Product ion spectra generated from the [M - 2H] 2- precursor ions were dominated by the loss of HSO 4 - to generate two product ions, that is, the ion at m/z 97 (HSO 4 - ) and the ion corresponding to the remaining monosulfate fragment. Other product ions were found to be specific for some structures. As an example, the loss of [CH 3 + SO 3 ] - was found to be important for several compounds with unsaturation adjacent to the sulfate. On the basis of the common behavior of the bis-sulfate metabolites two alternatives were evaluated for the untargeted detection of bis-sulfate metabolites (i) a precursor ion scan method using the ion at m/z 97 and (ii) a constant ion loss (CIL) method using the loss of HSO 4 - . Both methods allowed for the untargeted detection of the model compounds. Eight steroid bis-sulfates were synthesized in high purity in order to quantitatively evaluate the developed strategies. Lower limits of detection (2-20 ng/mL) were obtained using the CIL method. Additionally, the CIL method was found to be more specific in the detection of urinary bis-sulfates. The applicability of the CIL approach was demonstrated by determining progestogens altered during pregnancy and by detecting the bis-sulfate metabolites of tibolone.

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid-related compounds. Subsequent gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α- and 3β- isomers of the novel compound; 3-chloro-17α-methyl-5α-androstan-17β-ol. Synthesis of authentic reference materials followed by comparison of NMR, GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) data confirmed the finding of a new 'designer' steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α-chloro-17α-methyl-5α-androstan-17β-ol using equine and human S9 liver fractions were performed. For equine, GC-MS/MS analysis identified the diagnostic 3α-chloro-17α-methyl-5α-androstane-16α,17β-diol metabolite. For human, the 17α-methyl-5α-androstane-3α,17β-diol metabolite was found. Results from these studies were used to verify the ability of GC-MS/MS precursor-ion scanning techniques to support untargeted detection strategies for designer steroids in anti-doping analyses. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) is a designer anabolic androgenic steroid that is readily available via the internet. It contains an isoxazole fused to the steroid A-ring which offers metabolic stability and noteworthy anabolic activity raising concerns over the potential for abuse of this compound in equine sports. The metabolism of furazadrol was studied by in vivo and in vitro methods for the first time. Urinary furazadrol 17-sulfate and furazadrol 17-glucuronide metabolites were detected in vivo after a controlled administration and compared with synthetically-derived reference materials in order to confirm their identities. They were quantified to establish the excretion profile and a suitable limit of detection. Minor metabolites were also detected, including epifurazadrol, hydroxylated furazadrol, and hydroxylated and oxidised furazadrol, present as the sulfate and glucuronide conjugates. Phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli β-glucuronidase and Pseudomonas aeruginosa arylsulfatase to further confirm the identity of the corresponding phase I metabolites. The metabolism profile was compared to the products obtained from an in vitro phase I metabolism study, with all but two of the minor in vivo phase I metabolites observed in the in vitro system. These investigations identify the key urinary metabolites of furazadrol following oral administration, which can be incorporated into anti-doping screening and confirmation procedures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation., (Copyright © 2015 John Wiley & Sons, Ltd.)

Steroid sulfates are a major class of steroid metabolite that are of growing importance in fields such as anti-doping analysis, the detection of residues in agricultural produce or medicine. Despite this, many steroid sulfate reference materials may have limited or no availability hampering the development of analytical methods. We report simple protocols for the rapid synthesis and purification of steroid sulfates that are suitable for adoption by analytical laboratories. Central to this approach is the use of solid-phase extraction (SPE) for purification, a technique routinely used for sample preparation in analytical laboratories around the world. The sulfate conjugates of sixteen steroid compounds encompassing a wide range of steroid substitution patterns and configurations are prepared, including the previously unreported sulfate conjugates of the designer steroids furazadrol (17β-hydroxyandrostan[2,3-d]isoxazole), isofurazadrol (17β-hydroxyandrostan[3,2-c]isoxazole) and trenazone (17β-hydroxyestra-4,9-dien-3-one). Structural characterization data, together with NMR and mass spectra are reported for all steroid sulfates, often for the first time. The scope of this approach for small scale synthesis is highlighted by the sulfation of 1μg of testosterone (17β-hydroxyandrost-4-en-3-one) as monitored by liquid chromatography-mass spectrometry (LCMS)., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.

A gene coding for a protein containing two Scavenger Receptor Cysteine-Rich (SRCR) motifs, four Limulus factor C, Coch-5b2 and Lgl1 (LCCL) motifs; and one Polycystin-1, Lipoxygenase and Alpha Toxin (PLAT) motif was cloned from Plasmodium chabaudi and homologues identified in the P. falciparum and P. yoelii genome data bases. At least one of these sequence motifs (SRCR) has adhesive properties in other proteins, therefore, we propose to name this protein PSLAP for Plasmodium SRCR, LCCL Adhesive-like Protein. Southern blotting and chromosome analysis showed that pslap is a single copy gene on chromosome 14 in P. falciparum 3D7. pslap mRNA is strongly expressed in P. falciparum gametocytes, but was undetectable on Northern blots of RNA from the asexual blood stages. Polyclonal antibodies raised to different parts of PSLAP detected a protein expressed in late gametocytes, but not in the early stages of gametocytogenesis or asexual blood stages of P. falciparum. We suggest that PSLAP functions in the mosquito, for example, in modulation of the invertebrate host immune response or in protection against complement factors in the blood meal.