Catrin Goebel

The synthesis and pharmacological evaluation of adamantane-derived indoles: Cannabimimetic drugs of abuse

Two novel adamantane derivatives, adamantan-1-yl(1-pentyl-1H-indol-3-yl)methanone (AB-001) and N-(adamtan-1-yl)-1-pentyl-1H-indole-3-carboxamide (SDB-001), were recently identified as cannabimimetic indoles of abuse. Conflicting anecdotal reports of the psychoactivity of AB-001 in humans, and a complete dearth of information about the bioactivity of SDB-001, prompted the preparation of AB-001, SDB-001, and several analogues intended to explore preliminary structure-activity relationships within this class. This study sought to elucidate which structural features of AB-001, SDB-001, and their analogues govern the cannabimimetic potency of these chemotypes in vitro and in vivo. All compounds showed similar full agonist profiles at CB1 (EC50 = 16-43 nM) and CB2 (EC50 = 29-216 nM) receptors in vitro using a FLIPR membrane potential assay, with the exception of SDB-002, which demonstrated partial agonist activity at CB2 receptors. The activity of AB-001, AB-002, and SDB-001 in rats was compared to that of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) and cannabimimetic indole JWH-018 using biotelemetry. SDB-001 dose-dependently induced hypothermia and reduced heart rate (maximal dose 10 mg/kg) with potency comparable to that of Δ(9)-tetrahydrocannabinol (Δ(9)-THC, maximal dose 10 mg/kg), and lower than that of JWH-018 (maximal dose 3 mg/kg). Additionally, the changes in body temperature and heart rate affected by SDB-001 are of longer duration than those of Δ(9)-THC or JWH-018, suggesting a different pharmacokinetic profile. In contrast, AB-001, and its homologue, AB-002, did not produce significant hypothermic and bradycardic effects, even at relatively higher doses (up to 30 mg/kg), indicating greatly reduced potency compared to Δ(9)-THC, JWH-018, and SDB-001.

Studies of methylhexaneamine in supplements and geranium oil

A number of supplements are now available which are sold as fat burners or pre-workout boosters and contain stimulants which are banned in sport. Many contain methylhexaneamine under one of many pseudonyms including Geranamine, geranium oil or extract, or a number of chemical names such as 1,3-dimethylpentylamine. This has resulted in many athletes returning an adverse finding and having sanctions imposed. Other stimulants such as caffeine, phenpromethamine, synefrine, and phenethylamines are also to be found in supplements. This communication shows that geranium oils do not contain methylhexaneamine and that products labelled as containing geranium oil but which contain methylhexaneamine can only arise from the addition of synthetic material. Since the usual dose of methylhexaneamine is large, the drug is excreted at relatively high amounts for more than 29 h, the time for which the excretion was studied.

Effects of recombinant human LH and hCG on serum and urine LH and androgens in men

Context  The administration of gonadotrophins is prohibited in sport but the effect in men of recently available recombinant hCG and LH on serum and urine concentrations of gonadotrophins and androgens has not been systematically evaluated in the antidoping context.

Screening for testosterone abuse in male athletes using the measurement of urinary LH, a revision of the paradigm.

The primary screening method for the detection of doping by athletes using synthetic versions of endogenous steroids such as testosterone relies on measurement of the ratio of testosterone (T) to epitestosterone (E) in urine. In 2005 the World Anti-Doping Agency (WADA) lowered the T/E value at which samples undergo further investigation from six to four. This has resulted in a large increase in the number of athletes with naturally elevated T/E ratios undergoing investigation without a corresponding increase in the number of proven doping offences involving testosterone.Our objective was to develop a new simple screening protocol that can, with high probability, not only distinguish athletes whose natural T/E values exceed four from those whose T/E values have been elevated by testosterone doping but also detect those athletes with naturally low T/E values that do not exceed four despite being administered testosterone.Testosterone (250 mg Sustanon) was administered weekly to a group of 47 young adult males for five weeks in a double-blind placebo controlled study and urine samples collected. The samples were analysed for steroid concentrations using GC/MS and for luteinizing hormone (LH) by immunoassay.The elevation of T/E that occurred in all subjects was accompanied by a significant reduction in urinary LH concentrations to levels that are rare in normal subjects.The appropriate measurement of urinary LH, with the measurement of T/E values, can markedly improve the efficiency of detection of doping with testosterone by male athletes, particularly those who have low natural T/E ratios.

Detection and effects on serum and urine steroid and LH of repeated GnRH analog (leuprolide) stimulation.

Non-steroidal drugs that increase endogenous testosterone (T) may be used to exploit ergogenic effects of androgens in power sports. While superactive GnRH analog use is suspected, neither screening nor detection tests are developed. This study aimed to determine if (a) stimulation for 5 days by leuprolide (a superactive GnRH analog) of serum and urine steroids and urine LH is reproducible at a 2 week interval, (b) nandrolone decanoate (ND) co-administration masks responses to leuprolide administration, (c) performance of urine measurement of leuprolide and M1, its major metabolite, as a detection test. Healthy men were randomized into a 4 week parallel group, open label clinical study in which all men had daily sc injections of leuprolide (1mg) for 4 days in the 1st and 3rd weeks with hormone-free 2nd and 4th weeks. In the 3rd week, men were randomized to either ND injections or no extra treatment. Serum steroids were determined by liquid chromatography, tandem mass spectrometry (LC-MS), urine steroids by gas chromatography, mass spectrometry (GC-MS), urine leuprolide and M1 by high resolution LC-MS and urine LH by immunoassay. Leuprolide stimulated striking, reproducible increases in serum and urine LH and steroids (serum T, dihydroT (DHT), 3α diol; urine T, epitestosterone (E) and androsterone (A). ND suppressed basal serum T, E2, 3α diol, and urinary E but did not mask or change the magnitude of responses to leuprolide. Urine leuprolide and M1 measurement had 100% sensitivity and specificity in detecting leuprolide administration up to one day after cessation of injections with the detection window between 1 and 3 days after last dose. Screening using urine steroid and LH measurements, optimally by urinary log10(LHxT), correctly classified 82% of urine samples. It is concluded that leuprolide stimulation of endogenous testosterone is reproducible after a 10-day interval, is not masked by ND and is reliably detected by urine leuprolide or M1 measurement for at least 1 day after administration.

Carbon isotope ratio analysis of endogenous glucocorticoid urinary metabolites after cortisone acetate and adrenosterone administration for doping control

Glucocorticoids are listed on the World Anti-Doping Agency (WADA) Prohibited List of substances. The detection of the administration of hydrocortisone and cortisone is complicated by the fact that the human body also produces these steroids naturally. Gas chromatography-combustion-isotope ratio mass spectrometry can be utilized to determine the use of endogenous glucocorticoids by measuring the carbon isotope ratio (CIR) of their resulting metabolites in human urine samples. A comprehensive sample preparation protocol for the analysis of endogenous glucocorticoid urinary metabolites was developed and validated, incorporating the use of high performance liquid chromatography (HPLC) for purification and chemical oxidation for derivatisation. Target compounds were tetrahydrocortisol and tetrahydrocortisone, and 11β-hydroxyetiocholanolone, 11-oxoetiocholanolone and 11β-hydroxyandrosterone, while pregnanediol functioned as the endogenous reference compound. Urine samples from a population of 50 volunteers were analyzed to determine CIR reference limits. Excretion studies of the endogenous glucocorticoid preparation cortisone acetate (25 mg oral) and the dietary supplement adrenosterone (75 mg oral) were conducted with six male individuals. Variable changes in steroid metabolite isotopic composition were found across subjects after administration. The study also revealed that CIR analysis of the major glucocorticoid metabolites tetrahydrocortisol and tetrahydrocortisone is necessary to unambiguously distinguish administration of cortisone and adrenosterone, the former officially restricted to out-of-competition use by athletes, the latter not being restricted at the current time. Moreover, this study reaffirms that CIR methods for the doping control of endogenous steroids should not rely upon a single ERC, as the administration of an appropriate precursor to that ERC could cause complications during analysis.

Stimulating luteinizing hormone

Low molecular weight luteinizing hormone (LMWLH) receptor agonists could be of interest as a potential doping substance for athletes. These orally active compounds induce the production of endogenous hormones such as testosterone in a similar way to LH. A method for the detection of these compounds needs to be direct as their effect--the excess production of endogenous hormones--cannot be proven by analysis techniques which test for endogenous hormones. In order to detect a broad range of potential LMWLH receptor agonists, a precursor ion monitoring liquid chromatography tandem mass spectrometry method was developed. The method was tested against a selection of urine samples to ascertain potential problems with background analytes interfering with the compounds of interests. Selected compounds were extracted with an established methodology from urine to determine suitability of implementing into general screening methodologies. The two available LMWLH receptor agonists could be detected at concentrations of 100 ng/ml in urine samples. This establishes a basic precursor ion monitoring method suitable for screening purposes for the detection of LMWLH receptor agonists in urine samples.

DMSO assisted electrospray ionization for the detection of small peptide hormones in urine by dilute-and-shoot-liquid-chromatography-high resolution mass spectrometry

AbstractThe mobile phase additive (DMSO) has been described as a useful tool to enhance electrospray ionization (ESI) of peptides and proteins. So far, this technique has mainly been used in proteomic/peptide research, and its applicability in a routine clinical laboratory setting (i.e., doping control analysis) has not been described yet. This work provides a simple, easy to implement screening method for the detection of doping relevant small peptides (GHRPs, GnRHs, GHS, and vasopressin-analogues) with molecular weight less than 2 kDa applying DMSO in the mobile phase. The gain in sensitivity was sufficient to inject the urine samples after a 2-fold dilution step omitting a time consuming sample preparation. The employed analytical procedure was validated for the qualitative determination of 36 compounds, including 13 metabolites. The detection limits (LODs) ranged between 50 and 1000 pg/mL and were compliant with the 2 ng/mL minimum detection level required by the World Anti-Doping Agency (WADA) for all the target peptides. To demonstrate the feasibility of the work, urine samples obtained from patients who have been treated with desmopressin or leuprolide and urine samples that have been declared as adverse analytical findings were analyzed. Graphical Abstractᅟ