A screening method for the quantitative determination of selective androgen receptor modulators (SARMs) in capsules by high resolution 19F- and 1H-NMR spectroscopy.

A new method for rapid determination of the content of selective androgenic receptor modulators (SARMs) andarine, cardarine, ligandrol, ostarine and S-23 in capsules by 1H- and 19F-high resolution nuclear magnetic resonance spectroscopy was described and validated. Specificity, linearity, accuracy, precision, detection and quantification limits were considered as validation parameters. Full 1H-, 13C- and 19F-NMR structural assignment of the SARMs is provided as a tool for self-standing identification without a reference standard. Amounts of 7-15 mg of SARMs/capsule were detected in different products with an intermediate precision of 0.8-1.7% in 4 to 20 minutes of analysis time. The validation results and rapidity of analysis confirm the applicability of the method for large-scale screening. The statistical analysis of the results from 19F- and 1H-quantitative NMR showed that both approaches were equally effective, thus expanding the potential use of the methodology to non-fluorinated SARMs. At present, no SARM has been approved for human consumption; however, SARMs are actually used by bodybuilders and recreational athletes, who purchase them even though the risk-benefit ratio of these molecules has not been definitively established.

A review of current chemistry, pharmacology, and regulation of endogenous anabolic steroids testosterone, boldenone, and nandrolone in horses.

Anabolic androgenic steroids are synthetic substances related to the male sex hormones (androgens). These agents promote the growth of skeletal muscle (anabolic effects) and the development of male sexual characteristics (androgenic effects). Anabolic steroids have been illegally used for many years as performance-enhancing drugs in human, equine, and canine sports and as growth promoters in livestock reared to provide meat for human consumption. The analytical challenge to developing effective means of control within these fields has been exacerbated by the reported endogenous nature of some of these steroids. Anabolic steroids have been employed extensively in equine practice over the past 50 years. Their usefulness is largely dependent on subjective opinions, as only minimal studies investigating pharmacodynamics have been carried out in horses. Therefore, their use will vary markedly between practitioners depending on their personal experiences and pressures by trainers to use them. They form part of rational therapy in a variety of conditions. In addition to their use for increasing muscle mass, they are used to varying extents in the raising of yearlings and in the training and racing of horses with the view of improving performance. The use of these agents is prohibited in the horseracing industry by the Association of Racing Commissioners International (ARCI), International Federation of Horseracing Authorities (IFHA), and Fédération Equestre Internationale (FEI).

Detection of DHCMT long-term metabolite glucuronides with LC-MSMS as an alternative approach to conventional GC-MSMS analysis.

Dehydrochloromethyltestosterone (DHCMT) is one of the most detected illicit used anabolic-androgenic steroids in professional sports. Therefore, a fast and accurate analysis of this substance is of great importance for a constructive fight against doping abuse. The conventional method for the analysis of this drug, GC-MSMS, is very sensitive and selective but also very time- and resource-consuming. With the presented work, a new approach for simple detection with LC-HRMSMS without any sample preparation is introduced. The method is based on the direct analysis of two newly described phase-II metabolites of the DHCMT long-term metabolite 4-chloro-18-nor-17ß-hydroxymethyl-17α-methyl-5ß-androst-13-en-3α-ol (M3). LC-HRMSMS, GC-MSMS, fractionation and derivatization experiments are combined to identify and characterize for the first time two different glucuronide-acid conjugates of this metabolite in positive human urine samples. In addition, a third glucuronide metabolite was identified, however without isomeric structure determination. The detection of these metabolites is particularly interesting for confirmation analyses, as the method is rapid and requires little sample material.

Androgens, sports, and detection strategies for anabolic drug use.

For decades, anabolic androgenic agents have represented the substance class most frequently observed in doping control samples. They comprise synthetic and pseudoendogenous anabolic androgenic steroids and other, mostly non-steroidal compounds with (presumed) positive effects on muscle mass and function. While exogenous substances can easily be detected by gas/liquid chromatography and mass spectrometry, significantly more complex methodologies including the longitudinal monitoring of individual urinary steroid concentrations/ratios and isotope ratio mass spectrometry are required to provide evidence for the exogenous administration of endogenous compounds. This narrative review summarizes the efforts made within the last 5 years to further improve the detection of anabolic agents in doping control samples. Different approaches such as the identification of novel metabolites and biomarkers, the acquisition of complementary mass spectrometric data, and the development of new analytical strategies were employed to increase method sensitivity and retrospectivity while simultaneously reducing method complexity to facilitate a higher and faster sample throughput.

Discovery of 3-amino-4-{(3S)-3-[(2-ethoxyethoxy)methyl]piperidin-1-yl}thieno[2,3-b]pyridine-2-carboxamide (DS96432529): A potent and orally active bone anabolic agent.

The continuing investigation of SAR of 3-aminothieno[2,3-b]pyridine-2-carboxamide derivatives has been described. In this study, C4-piperidine derivatives with polar functional groups were synthesized to develop orally available bone anabolic agents. The optimized compound 9o (DS96432529), which exhibited the best PK profile and high in vitro activity, showed the highest in vivo efficacy in this series. Moreover, significant synergistic effects were observed following co-administration of DS96432529 and alendronate or parathyroid hormone. The mechanism of action is most likely mediated through CDK8 inhibition.

Liquid chromatography-mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes.

Intact phase II steroid metabolites have poor product ion mass spectra under collision-induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17ß-isomers of the sulfate conjugates, but limited differentiation for 17α/17ß-isomers of glucuronide conjugates and for 3α/3ß- and 5α/5ß-stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed-according to the World Anti-Doping Agency (WADA) TD2015IDCR-the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC-ESI-MS/(MS).

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Structurally different anabolic androgenic steroids reduce neurite outgrowth and neuronal viability in primary rat cortical cell cultures.

The illicit use of anabolic androgenic steroids (AAS) among adolescents and young adults is a major concern due to the unknown and unpredictable impact of AAS on the developing brain and the consequences of this on mental health, cognitive function and behaviour. The present study aimed to investigate the effects of supra-physiological doses of four structurally different AAS (testosterone, nandrolone, stanozolol and trenbolone) on neurite development and cell viability using an in vitro model of immature primary rat cortical cell cultures. A high-throughput screening image-based approach, measuring the neurite length and number of neurons, was used for the analysis of neurite outgrowth. In addition, cell viability and expression of the Tubb3 gene (encoding the protein beta-III tubulin) were investigated. Testosterone, nandrolone, and trenbolone elicited adverse effects on neurite outgrowth as deduced from an observed reduced neurite length per neuron. Trenbolone was the only AAS that reduced the cell viability as indicated by a decreased number of neurons and declined mitochondrial function. Moreover, trenbolone downregulated the Tubb3 mRNA expression. The adverse impact on neurite development was neither inhibited nor supressed by the selective androgen receptor (AR) antagonist, flutamide, suggesting that the observed effects result from another mechanism or mechanisms of action that are operating apart from AR activation. The results demonstrate a possible AAS-induced detrimental effect on neuronal development and regenerative functions. An impact on these events, that are essential mechanisms for maintaining normal brain function, could possibly contribute to behavioural alterations seen in AAS users.

Severe Persistent Jaundice after the Abuse of an Anabolic Androgenic Steroid Analogue.

Hepatic disorders with prominent cholestasis can be caused by a range of conditions, and anabolic androgenic steroids have been considered a cause of protracted cholestasis. A 29-year-old man who had taken an anabolic androgenic steroid analogue for 2 months visited the hospital complaining of jaundice and indigestion. After stopping the medication, the hyperbilirubinemia tended to decrease, but a transiently elevated aminotransferase level was observed. The endogenous testosterone level also decreased initially but recovered soon after. The liver function profiles were normalized after 2 months of conservative management. This case emphasizes that close drug history taking, including anabolic steroids, is important for identifying the cause of unexplained persistent jaundice.

Identification of osteogenic progenitor cell-targeted peptides that augment bone formation.

Activation and migration of endogenous mesenchymal stromal cells (MSCs) are critical for bone regeneration. Here, we report a combinational peptide screening strategy for rapid discovery of ligands that not only bind strongly to osteogenic progenitor cells (OPCs) but also stimulate osteogenic cell Akt signaling in those OPCs. Two lead compounds are discovered, YLL3 and YLL8, both of which increase osteoprogenitor osteogenic differentiation in vitro. When given to normal or osteopenic mice, the compounds increase mineral apposition rate, bone formation, bone mass, and bone strength, as well as expedite fracture repair through stimulated endogenous osteogenesis. When covalently conjugated to alendronate, YLLs acquire an additional function resulting in a "tri-functional" compound that: (i) binds to OPCs, (ii) targets bone, and (iii) induces "pro-survival" signal. These bone-targeted, osteogenic peptides are well suited for current tissue-specific therapeutic paradigms to augment the endogenous osteogenic cells for bone regeneration and the treatment of bone loss.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene steroids.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene unit of the long-term metabolites of 17-methylated androgenic anabolic steroids (AAS) is reported. It is based on a visible light-promoted radical decarboxylative alkynylation of steroidal redox-active ester. The developed method was used in synthesis of the long-term metabolite of AAS oxymesterone.

Achieving enhanced bone regeneration using monetite granules with bone anabolic drug conjugates (C3 and C6) in rat mandibular defects.

Bone grafting procedures are commonly used to manage bone defects in the craniofacial region. Monetite is an excellent biomaterial option for bone grafting, however, it is limited by lack of osteoinduction. Several molecules can be incorporated within the monetite matrix to promote bone regeneration. The aim was to investigate whether incorporating bone forming drug conjugates (C3 and C6) within monetite can improve their ability to regenerate bone in bone defects. Bilateral bone defects were created in the mandible of 24 Sprague-Dawley rats and were then packed with monetite control, monetite+C3 or monetite+C6. After 2 and 4 weeks, post-mortem samples were analyzed using microcomputed tomography, histology and back-scattered electron microscopy to calculate the percentages of bone formation and remaining graft material. At 2 and 4 weeks, monetite with C3 and C6 demonstrated higher bone formation than monetite control, while monetite+C6 had the highest bone formation percentage at 4 weeks. There were no significant differences in the remaining graft material between the groups at 2 or 4 weeks. Incorporating these anabolic drug conjugates within the degradable matrix of monetite present a promising bone graft alternative for bone regeneration and repair in orthopedic as well as oral and maxillofacial applications.

Development of a method using gas chromatography-mass spectrometry for profiling of oil-based androgenic anabolic steroid products.

A GC-MS based analytical method was developed for the profiling of oil-based AAS products using 15 organic constituents as target compounds. A total of 219 compounds were identified in 109 seized AAS products, among them 15 target compounds were selected. The selection was based on each compound's occurrence, reproducibility, and variance between products. The 15 target compounds did not include the active steroid itself, but only compounds found in the carrier oil. The subsequent method validation included assessment of specificity, linearity, precision, robustness and sample stability. The method was finally applied for the classification of a set of 27 seizures of AAS products supplied by the police. The classification was based on the Pearson correlation coefficient using pre-treated peak area data from the 15 target compounds. A successful classification was obtained, with only a small overlap between linked and unlinked samples. A 1% false-positive rate could be obtained at a threshold of 0.625 in terms of the Pearson distance. The present study thus demonstrates that it is possible to profile and classify AAS products with regard to a common origin. As the profiling method is not specific with regards to the steroid content, it may potentially be used to profile and compare other kinds of oil-based liquids.

The synthesis of 4-chloro-17ß-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol - Proposed long term metabolite (M4) of oralturinabol.

4-Chloro-17ß-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol is one of proposed long term metabolites of oralturinabol (anabolic androgenic steroid restricted in sport). The synthesis of 4-chloro-17ß-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol was achieved. Isomerisation of configuration of 13-carbon was used for construction of 17ß-hydroxymethyl-17α-methyl fragment. The proposed route of synthesis allows to obtain 3ß-hydroxy isomer as well.

Design, synthesis and characterization of a PEGylated stanozolol for potential therapeutic applications.

Stanozolol (STZ) is a drug used to treat serious disorders like aplastic anemia and hereditary angioedema. It is also indicated as an adjunct therapy for the treatment of vascular disorders and growth failures. Encouraging results obtained using animal models demonstrated that STZ increases bone formation and mineralization, thus improving both density and biomechanical properties. Like natural androgens, such as TST and 5α-dihydrotestosterone (5α-DHT), STZ binds androgen receptor (AR) to activate AR-mediated signaling. Despite its therapeutic effects, this synthetic anabolic-androgenic steroid (AAS), or 5α-DHT derivative, due to its high lipophilicity, is poor soluble in water. Thus, to increase the water solubility and stability of STZ, as well as its bioavailability and efficacy, an innovative PEGylated STZ (STZ conjugated with (MeO-PEG-NH2)10kDa, (MeO-PEG-NH)10kDa-STZ) was synthesized. As confirmed by chromatography (RP-HPLC) and spectrometry (ATR-FTIR, 1H NMR, elemental CHNS(O) analysis, MALDI-TOF/TOF) analyses, a very pure, stable and soluble compound was obtained. Acetylcholinesterase (AChE) competitive ELISA demonstrated that the resulting PEGylated STZ competes against biological TST, especially at lower concentrations. Cytotoxicity of increasing concentrations (1, 10, 25 or 50 µM) of STZ and/or (MeO-PEG-NH)10kDa-STZ was also evaluated for up 80 h by performing the MTT assay on human osteosarcoma Saos-2 cells, which express AR and are responsive to STZ. PEGylation mitigated cytotoxicity of STZ, by increasing the cell viability values, especially at higher drug concentrations. Furthermore, these results suggest that (MeO-PEG-NH)10kDa-STZ is a promising and reliable drug to be used in clinical conditions in which TST is required.

A rapid and eco-friendly method for determination of the main components of gamma-oryzanol in equestrian dietary and nutritional supplements by liquid chromatography-Tandem mass spectrometry.

Gamma-oryzanol (GO) has gained special attention in the equine sports industry in recent years due to its touted properties, including the fact that it may cause anabolic effects on muscle growth and reduce fatigue. Many manufactures offer supplements containing GO as a naturally occurring anabolic substance; however, some producers do not declare its presence in product compositions. Taking into consideration the touted properties of GO, its ambiguous effectiveness and the open character of the Prohibited Substances List established by the Fédération Equestre Internationale, there is an urgent need to elaborate procedures for the estimation of horse exposure to GO during supplementation, as well as during routine analysis of supplements. This work describes the development and validation of the method for determination of the four main GO components, i.e., cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesteryl ferulate and ß-sitosteryl ferulate, in equestrian supplements based on LC-MS/MS after a simple ultrasound-assisted extraction (Eco-Scale score value of 76). The analytical performance achieved satisfactory results in terms of linearity (R2 > 0.9910), sensitivity (LODs ranged from 0.4 to 1.9 ng/mL), intra- and interday accuracy (from 90.4-115.8%), precision (CV < 9.6%) and recovery (from 87.6-108.6%) for all of the investigated compounds. The method was successfully applied to the analysis of thirty equestrian supplements.

Pharmacological Characterization of a Novel 5-Hydroxybenzothiazolone-Derived ß 2-Adrenoceptor Agonist with Functional Selectivity for Anabolic Effects on Skeletal Muscle Resulting in a Wider Cardiovascular Safety Window in Preclinical Studies.

The anabolic effects of ß 2-adrenoceptor (ß 2-AR) agonists on skeletal muscle have been demonstrated in various species. However, the clinical use of ß 2-AR agonists for skeletal muscle wasting conditions has been limited by their undesired cardiovascular effects. Here, we describe the preclinical pharmacological profile of a novel 5-hydroxybenzothiazolone (5-HOB) derived ß 2-AR agonist in comparison with formoterol as a representative ß 2-AR agonist that have been well characterized. In vitro, 5-HOB has nanomolar affinity for the human ß 2-AR and selectivity over the ß 1-AR and ß 3-AR. 5-HOB also shows potent agonistic activity at the ß 2-AR in primary skeletal muscle myotubes and induces hypertrophy of skeletal muscle myotubes. Compared with formoterol, 5-HOB demonstrates comparable full-agonist activity on cAMP production in skeletal muscle cells and skeletal muscle tissue-derived membranes. In contrast, a greatly reduced intrinsic activity was determined in cardiomyocytes and cell membranes prepared from the rat heart. In addition, 5-HOB shows weak effects on chronotropy, inotropy, and vascular relaxation compared with formoterol. In vivo, 5-HOB significantly increases hind limb muscle weight in rats with attenuated effects on heart weight and ejection fraction, unlike formoterol. Furthermore, changes in cardiovascular parameters after bolus subcutaneous treatment in rats and rhesus monkeys are significantly lower with 5-HOB compared with formoterol. In conclusion, the pharmacological profile of 5-HOB indicates superior tissue selectivity compared with the conventional ß 2-AR agonist formoterol in preclinical studies and supports the notion that such tissue-selective agonists should be investigated for the safe treatment of muscle-wasting conditions without cardiovascular limiting effects.

Doping substances in dietary supplements. / Dopingmidler i kosttilskudd.

BACKGROUND: International studies have shown that 12-58 % of all dietary supplements intended for people who exercise and engage in sports contain substances prohibited by the World Anti-Doping Code (WADC). In some cases, the doping substances are not declared on the product label, and the consumer may therefore be unaware of what he/she ingests. Many of the substances may cause adverse health effects, and sale of such products is illegal in Norway. MATERIAL AND METHOD: To investigate the prevalence of doping substances in dietary supplements sold on the Norwegian market, a total of 93 high-risk products from online shops targeting Norwegian consumers were analysed for substances on the WADC Prohibited List and pharmaceutical drugs. All supplements were marketed as able to boost energy levels and/or having a muscle-building or fat-burning effect. The products were selected on the basis of tips received, online forums and/or international lists. RESULTS: Altogether 21 of 93 (23 %) products analysed contained prohibited substances, pharmaceutical drugs and/or illegal amounts of caffeine. Substances on the WADC Prohibited List were detected in 8 of the 93 (9 %) dietary supplements. All products containing doping substances were declared as containing one or more banned substances. INTERPRETATION: The results show that using apparently legal dietary supplements purchased in online shops targeting Norwegian consumers involves a risk of inadvertent doping and adverse health effects.

Application of differential scanning calorimetry in the analysis of apprehended formulations of anabolic androgenic steroids.

Over the past ten years, there has been a significant increase in the amount of formulations containing anabolic androgenic steroids apprehended worldwide. A considerable amount of these illicit preparations is falsified imposing a series of challenges for the analytical identification of alleged active ingredients due to the presence of interferers. In this sense, the aim of this work was to identify and quantify the active ingredient using cholesterol as internal standard in eight apprehended formulations of anabolic androgenic steroids in either tablet, capsule or injectable forms employing visual inspection and instrumental analysis of Fourier Transform Infrared Spectroscopy, Gas Chromatography - Mass Spectrometry and Differential Scanning Calorimetry. The assessed samples were kindly provided by the Brazilian Federal Police as representative samples from an apprehension made in July of 2017. Qualitatively, 25% of the analyzed materials were determined to be falsified as they were composed of excipients only while the others had the alleged active ingredient confirmed. However, after quantitative analysis, the majority of samples were placed as counterfeit materials as the active substance was found in concentrations lower than stated in the label. Preliminary visual inspection provided important information to distinguish genuine from falsified samples. It should be noted that this work was one of the few available reports to employ Differential Scanning Calorimetry in the analysis of anabolic agents, which proved to be an important complementary tool for the detection of the active ingredient, when present, along with the calorimetric profile of the formulations studied. Fourier Transform Infrared Spectroscopy and Gas-Chromatography - Mass Spectrometry were also efficient analytical tools in order to identify and to characterize substances present in fraudulent preparations.