Dehydrochloromethyltestosterone Research Articles

Detectability of oxandrolone, metandienone, clostebol and dehydrochloromethyltestosterone in urine after transdermal application.

Situations of both, intentional and inadvertent or accidental doping, necessitate consideration in today's doping controls, especially in the light of the substantial consequences that athletes are facing in case of so-called adverse analytical findings. The aim of this study was to investigate, whether a transdermal uptake of doping substances would be possible. In addition to the period of detectability of the particular substances or respective characteristic metabolites, the possibility of deducing the route of administration by metabolite patterns was also assessed. Twelve male subjects were included in the study. Four common anabolic androgenic steroids (AAS) were dissolved in dimethylsulfoxide to facilitate transdermal administration on different skin regions. One half of the test persons received only oxandrolone (17α-methyl-2-oxa-4,5α-dihydrotestosterone), and the other half were applied a mixture of oxandrolone, metandienone (17β-hydroxy-17α-methylandrosta-1,4-dien-3-one), clostebol (4-chlorotestosterone-17β-acetate) and dehydrochloromethyltestosterone (DHCMT). Urine samples were collected 1 h, 6 h and one sample per day for the next 14 consecutive days. Measurements were conducted on a tandem-gas chromatography-mass spectrometry (GC-MS/MS) or tandem-liquid chromatography-MS/MS (LC-MS/MS) system. Substance findings were obtained at least 1 day after application on nearly all skin locations. The results indicated inter-individual variability in detection windows, also varying between the different analytes and possible impact of skin location and skin thickness, respectively. Nevertheless, a rapid and rather long detectability of all substances (or respective metabolites) was given, in some cases within hours after administration and for up to 10-14 days. Hence, the transdermal application or exposure to the investigated AAS is a plausible scenario that warrants consideration in anti-doping.

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-17β-hydroxymethyl-17α-methyl-5β-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.

Organ-on-a-chip: Determine feasibility of a human liver microphysiological model to assess long-term steroid metabolites in sports drug testing.

A fundamental challenge in preventive doping research is the study of metabolic pathways of substances banned in sport. However, the pharmacological predictions obtained by conventional in vitro or in vivo animal studies are occasionally of limited transferability to humans according to an inability of in vitro models to mimic higher order system physiology or due to various species-specific differences using animal models. A more recently established technology for simulating human physiology is the "organ-on-a-chip" principle. In a multichannel microfluidic cell culture chip, 3-dimensional tissue spheroids, which can constitute artificial and interconnected microscale organs, imitate principles of the human physiology. The objective of this study was to determine if the technology is suitable to adequately predict metabolic profiles of prohibited substances in sport. As model compounds, the frequently misused anabolic steroids, stanozolol and dehydrochloromethyltestosterone (DHCMT) were subjected to human liver spheroids in microfluidic cell culture chips. The metabolite patterns produced and circulating in the chip media were then assessed by LC-HRMS/(MS) at different time points of up to 14 days of incubation at 37°C. The overall profile of observed glucurono-conjugated stanozolol metabolites excellently matched the commonly found urinary pattern of metabolites, including 3'OH-stanozolol-glucuronide and stanozolol-N-glucuronides. Similarly, but to a lower extent, the DHCMT metabolic profile was in agreement with phase-I and phase-II biotransformation products regularly seen in postadministration urine specimens. In conclusion, this pilot study indicates that the "organ-on-a-chip" technology provides a high degree of conformity with traditional human oral administration studies, providing a promising approach for metabolic profiling in sports drug testing.

Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail.Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite.The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite.It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

Metabolism of oral turinabol by the human brain cholesterol 24-hydroxylase CYP46A1

The human microsomal cytochrome P450 enzyme CYP46A1 plays a crucial role in cholesterol elimination from the brain. It performs a 24-hydroxylation of cholesterol and is of outstanding significance for memory and cognition. This study demonstrates the catalytic activity of human CYP46A1 towards an anabolic androgenic steroid, oral turinabol (dehydrochloromethyltestosterone, 4-chloro-17β-dihydroxy,17α-methylandrosta-1,4-dien-3-one), which is a doping substance. CYP46A1 is the first human microsomal steroid-converting P450 showing activity towards this xenobiotic compound. Furthermore, the inhibitory effect of oral turinabol on the cholesterol conversion has been investigated in vitro demonstrating competition of the two substrates on the active site of CYP46A1 which might be of importance for potential pathogenic effects of oral turinabol. The conversion of oral turinabol was found to be selective resulting in the formation of only one product, as shown by HPLC analysis. To produce sufficient amounts of this product for NMR analysis, a system expressing human full-length CYP46A1 and CPR on a bicistronic vector was successfully developed realizing the selective cholesterol 24-hydroxylation in E. coli in mg amounts. Using this novel whole-cell system, the conversion of oral turinabol was performed and the product of this conversion by CYP46A1 was isolated and identified as 16β-hydroxy oral turinabol by NMR.

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-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-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.

Synthesis of human long-term metabolites of dehydrochloromethyltestosterone and oxymesterone

We herein report the synthesis of the long-term metabolites “M4” (IUPAC: 4-chloro-17-hydroxymethyl-17-methyl-18-norandrosta-4,13-dien-3-ol) of dehydrochloromethyl-testosterone (DHCMT, Oral Turinabol) and “Oxy M9” (4-hydroxy-17β-hydroxymethyl-17α-methyl-18-norandrosta-4,13-dien-3-one) of oxymesterone (Oranabol). Both compounds were derived from a common synthetic route starting from dehydroepiandrosterone acetate. Four different stereoisomers were evaluated for metabolite M4. The previously assigned structure could be corrected regarding the C-3 and C-17 stereocenters.

Isomers of halodrol: synthesis and partial reduction to form A-ring of a long-term metabolite of dehydrochloromethyltestosterone (oral turinabol)

A new synthetic route to individual isomers of 4-chloro-17α-methylandrost-1,4-diene-3,17β-diol (halodrol, CDMA) based on reduction of the carbonyl group in dehydrochloromethyltestosterone (oral turinabol, DHCMT), followed by separation of epimers by fractional crystallisation from dichloromethane is presented. Reduction of the disubstituted double bond of the diene system in 3α-hydroxy isomer of halodrol afforded 4-chloro-17α-methyl-androst-4-ene-3α,17β-diol contained A-ring corresponding to 4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-4,13-dien-3α-ol (M4), which is one of the long-term metabolites of oral turinabol.

Unambiguous identification and characterization of a long-term human metabolite of dehydrochloromethyltestosterone.

In doping control analysis, the characterization of urinary steroid metabolites is of high interest for a targeted and long-term detection of prohibited anabolic androgenic steroids (AAS). In this work, the structure of a long-term metabolite of dehydrochloromethyltestosterone (DHCMT) was elucidated. Altogether, 8 possible metabolites with a 17α-methyl-17β-hydroxymethyl - structures were synthesized and compared to a major DHCMT long-term metabolite detected in reference urine excretion samples. The confirmed structure of the metabolite was 4α-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5α-androst-13-en-3α-ol.

Combined chemical and biotechnological production of 20βOH-NorDHCMT, a long-term metabolite of Oral-Turinabol (DHCMT)

Anabolic androgenic steroids (AAS) are misused very frequently in sport competitions as performance enhancing agents. One of the doping compounds that has been detected with increased frequency in the last few years is dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy-17α-methylandrosta-1,4-dien-3-one; brand name Oral Turinabol). The long-term DHCMT metabolite 20βOH-NorDHCMT (4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one) was reported earlier to be detectable in urine samples for more than 22 days after DHCMT administration; however, purified reference material was not available so far. In this study we demonstrate a successful combination of Wagner-Meerwein rearrangement of DHCMT to NorDHCMT (4-chloro-17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one) and subsequent whole-cell biotransformation with a recombinant fission yeast strain expressing the human cytochrome P450 enzyme (CYP or P450) CYP21A2 for the synthesis of mg amounts of this metabolite. It was then used as reference for the analysis of a post administration urine of DHCMT. The availability of this reference compound will provide an incontestable proof for DHCMT abuse.

Synthesis and structural elucidation of a dehydrochloromethyltestosterone metabolite.

The human urinary long-term metabolite "M3" (4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrost-13-en-3-ol) of the common doping agent DHCMT has thus far been detected via GC/MS-MS, creating ambiguities concerning its absolute configuration. Its structure was elucidated via the synthesis of all eight possible stereoisomers with 17β-hydroxymethyl configuration. The highlights of the synthesis consist of a novel first generation approach to 4β-chloro-5β compounds as well as a divergent route which allows easy access to the remaining A-ring chlorohydrins.

Critical Assessment of the Current WADA Approach for the Detection of 4-Chlorodehydromethyltestosteron

This paper is focused on application of method for detection of novel metabolites of 4-chlorodehydromethyltestosterone (known as oral turinabol, OT), which were postulated as long-term metabolites. The method started to be applied without rigorous validation, verification and excretion assay assisted as evidences for existence of the declared detectable compounds as true metabolites derived after OT biotransformation. This method has been started to use almost in its original form which arose in 2012 year for the very first time and never been revised and re-confirmed in peer-reviewed journals. Usually WADA encourages accredited laboratories to publish their results of methods development, validation and specific excretion studies in peer-reviewed journals. However, an incorrect method with neglected validation design and implicit data contradictions is currently widely applied. The author of this presented paper was athletes’ representative on repeated occasion and has been provided with several full documentation packages of OT novel metabolites analysis. Thus, the author makes its own conclusion based on its own evidences of that currently employed method and data analysis is exactly the same declared in a wrong way as emerged from the original paper.

Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes.

The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco- and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17β-hydroxy-17α-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11β-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11β,18-diOH-OT and 11β-OH-OT-18-al, which rearranges to its tautomeric form 11β,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies.

Cardiotoxicity of anabolic steroids; animal case study of oral turinabol and methanabol

Cardiotoxicity of anabolic steroids; animal case study of oral turinabol and methanabol

Identification of Potentially Counterfeit Ingredient in 4-Chlorodehydromethyl Testosterone Tablets by LC-ESI-MS

Identification of Potentially Counterfeit Ingredient in 4-Chlorodehydromethyl Testosterone Tablets by LC-ESI-MS

GC and capillary column GC/MS determination of synthetic anabolic steroids. II. 4-chloro-methandienone (oral turinabol) and its metabolites.

The determination of oral turinabol (4-chloro-17 alpha-methyl-17 beta-hydroxy-1,4-androstadien-3-one) [1] in the 'free' fraction of human urine samples by gas chromatography and capillary column gas chromatography/mass spectrometry was studied. After administration to man, three major metabolites are formed whose structures were identified as 6 beta-hydroxy-turinabol [2], 6 beta, 12-dihydroxy-turinabol [4], and 6 beta, 16-dihydroxy-turinabol [5], respectively. In much smaller quantities at least another three metabolites are excreted, one of which could be identified as 17 epi-turinabol [6]. No measurable amounts of 1 itself were detected in any of the urine samples investigated. The rate of metabolism and urinary excretion is reasonably fast. The total amount of recovered material, in the form of the three main metabolites, is on the order of 15%. Clean-up procedures and chromatographic conditions are presented in detail.

Stoffwechsel von steroidpharmaka: VI. Metaboliten von 4-chlor-17α-methyl-17β-hydroxy-1,4-androstadien-3-on durch mikrobielle hydrierung

The anabolic steroid Oral-Turinabol (4-chloro-17α-methyl-17β-hydroxy-1,4-androstadien-3-one) was hydrogenated with Clostridium paraputrificum and Rhodotorula glutinis. These micro-organisms stereospecifically reduce Δ 4-3-ketosteroids to 5β/3α- and 5α/3α-hydroxy-derivatives respectively. C1. paraputrificum transformed Oral Turinabol to 4β-chloro-17α-methyl-17β-hydroxy-5β-1-androsten-3-one and 4β-chloro-17α-methyl-5β-1-androsten-3α,17β-diol; Rh. glutinis to 4α-chloro-17α-methyl-17β-hydroxy-5α-1-androsten-3-one and 4α-chloro-17α-methyl-5α-1-androstene-3β, 17β-diol. In human beings the hydrogenation of Oral Turinabol is markedly inhibited. The microbiological hydrogenation is also diminished.

Stoffwechsel von steroidpharmaka: VII. Metaboliten von 4-chlor-17α-methyl-17β-hydroxy-1,4-androstadien-3-on durch mikrobielle hydroxylierung

The anabolic steroid Oral Turinabol (4-chloro-17α-methyl-17β-hydroxy-1,4-androstadien-3-one) was hydroxylated microbiologically. With Absidia glauca and Cunninghamella echinulata the 6β- and 7β- and with Aspergillus flavus the 15α- and 15β-hydroxyderivatives were obtained, In this way it is possible to prepare metabolites of Oral Turinabol which are needed for studies in metabolism and biological activity.