Projets de recherche

Code: 15A13CB 

The use of small peptides as growth hormones releasing factors has become increasingly popular in the last few years. As described in the literature, a whole set of small peptides emerged these last decades representing today a more and more attractive class of performance-enhancing drugs for amateur and professional athletes.  Several studies have reported the presence of these compounds in recent seized products confirming the illegal circulation of this class of substances. 
The best analytical tool to detect these small peptides and also their analogs or discover unknown similar compounds is the high resolution / high accuracy mass spectrometry (LC-HRMS). 
The aim of this study is to implement a high performance screening method by LC-HRMS in order to detect this new drug class. This project will focus on the available matrix from anti-doping tests: urine and blood (serum and plasma) but also on the seized products or internet-based drugs, which are the awareness keys of doping trends. Moreover, this project will also focus on the computer assisted method available with the LC-HRMS equipments. All the acquisition mode will be tested on the two technologies available in our laboratory (Q-Orbitrap and QqTOF) and an assessment of software helping to characterize metabolites without the reference material will be performed. The best strategy will be validated and applied to real samples from anti-doping controls.

Main Findings: 

A high performance screening procedure by LC-HRMS for prohibited peptides was developed and implemented in the laboratory. A protocol has been developed for urinary samples but also for plasma samples. Tests carried out show that the plasma method could also be easily applied to the serum samples. This screening protocol allows the detection of peptides < 2kDa but also peptides > 2kDa in a same method. The LOD obtained meet the WADA technical documents in force apart for larger peptides such as Tesamorelin, CJC-1295 or Sermorelin. For this category of peptides, another protocol should be applied in order to reach the expected LOD (0.5 ng/mL).  During this study, two kinds of instruments were evaluated: the QExactive+ system and the QTOF. The performances of the two instruments were globally equivalent but the QExactive+ instrument shows most of the time slightly better LODs and less background noise for some ion transitions. It was so retained as instrument of choice for drug testing routine analyses.  A stability study was performed during the validation process confirming that samples should be frozen as soon as possible after collection and stored frozen. A storage at +4°C is possible but should not exceed few days (3 days maximum for Alexamorelin or Goserelin for example).Seized products and internet-based compounds were also analyzed and characterized thanks to comparison with a database build on the instrument. On the twelve internet/seized products at our disposal, three of them showed incorrect identification on the original vial. For example, one vial identified as HGH frag 176-191 contains exclusively Sermorelin and another vial identified as TB-500 was in fact Thymosin 4. The last case was a vial identified as TB-1000 (supposed to be Thymosin 4) but it contains a mixture of TB-500 and its metabolite TB-500 M3.

Code: 15A15RV 

Current screening methods for exogenous anabolic androgenic steroids (AAS) are based on the hydrolysis of the urine samples using β-glucuronidase enzymes and the measurement of the released phase I metabolites using gas and liquid chromatography coupled to mass spectrometric techniques. In recent years, new long-term phase II metabolites of AAS, not detectable using the current screening conditions, have been identified using liquid chromatography-mass spectrometry technology, including glucuronoconjugated metabolites not readily hydrolysed with β-glucuronidase enzymes and sulfoconjugated metabolites. 
The hypothesis of the project is that significant information is missed in current AAS screening methods and the aim of the project is to develop a screening method based on the direct detection of the metabolites excreted in the urine, including phase II metabolites (glucuronides and sulfates) and relevant unconjugated metabolites using liquid chromatography coupled to tandem mass spectrometry with high resolution instruments. The method will improve the detection capabilities of AAS through the monitoring of important long-term metabolites, not detectable using the currently used screening methods. The approach will also facilitate the incorporation of new phase II metabolites. In addition, the possibility of acquiring full scan data will allow the retrospective reprocessing of all results for the detection of unknown metabolites, without the need of repeating the analysis. Moreover, the successful outcome of the project will represent a cost-effective simple approach, that can readily be shared amongst WADA accredited laboratories.

Main Findings: 

Current screening methods for exogenous anabolic androgenic steroids (AAS) are based on the hydrolysis of the urine samples using β-glucuronidase enzymes and the measurement of the released phase I metabolites using gas and liquid chromatography coupled to mass spectrometric techniques. In recent years, new long-term phase II metabolites of AAS, not detectable using the current screening conditions, have been identified using liquid chromatography-mass spectrometry technology, including glucuronoconjugated metabolites not readily hydrolysed with β-glucuronidase enzymes and sulfoconjugated metabolites.

The hypothesis of the project is that significant information is missed in current AAS screening methods and the aim of the project is to develop a screening method based on the direct detection of the metabolites excreted in the urine, including phase II metabolites (glucuronides and sulfates) and relevant unconjugated metabolites using liquid chromatography coupled to tandem mass spectrometry with high resolution instruments. The method will improve the detection capabilities of AAS through the monitoring of important long-term metabolites, not detectable using the currently used screening methods. The approach will also facilitate the incorporation of new phase II metabolites. In addition, the possibility of acquiring full scan data will allow the retrospective reprocessing of all results for the detection of unknown metabolites, without the need of repeating the analysis. Moreover, the successful outcome of the project will represent a cost-effective simple approach, that can readily be shared amongst WADA accredited laboratories.
         

Results and Conclusions:

Anabolic androgenic steroids (AAS) are the most frequently reported group of prohibited substances detected in doping controls. In recent years, new phase II metabolites of these compounds have been identified by LC-MS/MS and their usefulness improving the detection capabilities of the AAS misuse was demonstrated. However, the detection of some analytes was compromised due to the co-elution of endogenous interferences sharing the same ion transitions. The aim of this work was to evaluate the possibility of high-resolution mass spectrometry (HRMS), performed with LC-Q-TOF MS and LC-Q-Orbitrap MS systems, to overcome these limitations, thanks to the increased selectivity provided by the determination of the exact mass.  

The study of the ionization and fragmentation behaviour of unconjugated, sulfates and glucuronides steroids was performed with both Q-TOF and Q-Orbitrap instruments, acquiring fullscan and product ion spectra at different collision energies in positive and negative electrospray modes. The chromatographic conditions were optimized to achieve the best baseline separation of the isobaric compounds and to limit matrix interferences. The LC separation was finally performed with an Acquity UPLC BEH C18 column using H2O and CH3CN:H2O 95:5 (v/v) (0.01% formic acid, 1 mM ammonium formate) as mobile phase, with a total run time of 23 min. Solid-phase extraction (SPE) with Bond Elut C18 cartridges was used for sample clean-up and pre-concentration. Limits of detection (LODs), estimated with both HRMS systems, showed values below the 50% of the MRPL for sulfates and most of the glucuronides tested. Lower ionization efficiencies were obtained for some unconjugated steroids, producing unsatisfactory LODs with respect to WADA requirements.  

The Q-TOF-HRMS method was applied to urine samples obtained from oral and/or intramuscular administration of stanozolol, clostebol, methyltestosterone and metandienone, with the purpose to include in the study markers of these compounds that had been identified in previous works and for which standards were not available. Most of the main metabolites were identified on the basis of the accurate mass, relative retention time and characteristic fragmentation.  

Metabolomics strategies and statistical data analysis were used with the excretion studies after stanozolol and clostebol administrations to try to identify new metabolites. The most important metabolites already reported in the literature were identified using PLS-DA, but unfortunately, no new metabolites were discovered. Nevertheless, the results demonstrate the usefulness of the metabolomics strategy for the study of new metabolites. 
 
Overall, non-targeted screening methods based on HRMS instrumentation are a very useful tool in routine anti-doping analysis, with comparable applicability with respect to targeted MRM methods.

Code: 15A04JM 

The aim of the project is to produce a replacement freeze-dried human urine matrix certified reference material for the six steroids specified in the WADA technical document TD2014EAAS as markers for the urinary steroid profile. The material will be certified to a high level of accuracy with low uncertainties for the mass fraction and concentration of the steroid markers and testosterone to epitestosterone (T/E) ratio. The certified values will be assigned using isotope-dilution mass spectrometry and will be traceable to the International System of Units (SI). This material is intended to replace the stock of certified reference material NMIA MX005 which will be depleted over the next two years. MX005 has been used by WADA laboratories since its release in 2009 to demonstrate comparability of measurements now used for the Athlete Biological Passport (ABP). The ABP benefits all athletes by improving detection of doping and benefits individuals with unusual profiles by reducing unjustified scrutiny. The success of the ABP relies on results of
analysis for all samples from an individual athlete being comparable irrespective of the WADA laboratory that performed the analysis.  Comparability can be demonstrated by the appropriate use of a CRM specifically designed for the purpose.

Main Findings: 

A new freeze-dried human urine matrix certified reference material (CRM) has been produced to assist laboratories in longitudinal profiling measurements used for the Athlete Biological Passport and in the detection of testosterone abuse. Approximately 2400 units of the CRM (designated NMIA MX017) were produced and are now available to WADA accredited laboratories.  The material is provided with SI traceable reference values for the mass fractions and mass concentrations of the glucuronides of six steroids specified in the WADA technical document TD2018EAAS as markers for the urinary steroid profile: testosterone (T), epitestosterone (E), androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α17β-diol. 

The CRM was produced using pooled urine from six healthy individuals that had been fortified with testosterone glucuronide to provide a T/E mass ratio of 4. The mass fractions and concentrations of the target steroid glucuronides in the reconstituted urine were determined using the primary ratio analytical method of exact matching double isotope dilution mass spectrometry (IDMS) following extensive sample clean-up. The analytical procedure developed and optimised for this CRM employed a heart-cutting two dimensional HPLC purification of the target analytes and quantification using five characteristic molecular fragments in tandem mass spectrometry or high resolution mass spectrometry coupled with two different GC columns.

Estimates of the uncertainty in the certified values are based on investigation into all sources of uncertainty in inputs to the measurement equation and on studies of the homogeneity and stability of the mass fractions of the six steroid metabolites in the freeze-dried urine material during long-term storage, transport and use. Mass fraction and concentration reference values in the Certificate of Analysis for the CRM have expanded uncertainties (at the 95% level of confidence) below 5%. This is the same or better than previous similar CRMs, ensuring that NMIA MX017 will be a suitable replacement in controlling the quality of longitudinal profiling data.

Code: ISF15A35PV 

Sewage based tracing of doping use by amateur athletes. The present project investigates the applicablilty of sewage analysis to assess doping use in the general population and for amateur athletes in particular. To this end an analytical chemical tool will be developed that can identify and quantify existing as well as new doping substances present in sewage and pooled urine. The project will assess uncertainties associated with the various steps of sewage based epidemiology: sampling, sewage treatment plant and sewer system design, excretion rates, substance stability (in sewer), population size. The methodology used will employ high resolution mass spectrometry that will allow identification of hitherto unknown doping substances, quantification of known substances, and retrospective searches for newly identified doping agents in samples earlier collected. The methodology developed will be tested in sewage from three major cities in The Netherlands and during a major amateur sport event.

Main Findings: 

The present study investigated the applicability of the chemical analysis of wastewater to assess the use of doping substances by the general population and amateur athletes. To this end, an analytical methodology that can identify and quantify a list of substances from the groups of anabolic steroids, weight-loss products and masking agents in wastewater has been developed. The method uses solid phase extraction to increase the detection sensitivity of the target analytes, expected to be present at very low concentrations (ng L-1 range), and decrease possible matrix interferences. Instrumental analysis is performed by liquid chromatography coupled to high-resolution mass spectrometry, allowing data acquisition in both full scan and tandem MS mode. The method has been successfully validated at two concentration levels (50 and 200 ng L-1) with limits of quantification ranging between 0.7 and 60 ng L-1, intra- and inter-day precision expressed as relative standard deviation below 15%, procedural recoveries between 60 and 160% and matrix effects ranging from 45 to 121%. The stability of the analytes in wastewater was evaluated at different storage temperatures illustrating the importance of freezing the samples immediately after collection. The application of the method to 24-h composite wastewater samples collected at the entrance of three wastewater treatment plants and one pumping station prior to, during, and immediately after different sport events revealed the presence in wastewater, and hence the use, of the weight-loss substances ephedrine, norephedrine, methylhexanamine and 2,4-dinitrophenol. In particular norephedrine and 2,4-dinitrophenol use appeared to coincide with the event., and in greater quantities than anabolic steroids. 

Code: 15A41CR 

The detection of the misuse of these peptides is challenging due to a rapid elimination and consequently low concentrations in body fluids. Despite the analytical challenge, screening procedures have already been established, targeting numerous GHRPs in urine, employing liquid chromatographic/mass spectrometric detection. Thus, the focus has mainly been on the detection of the parent compounds. The additional incorporation of metabolites in routine detection methods would improve detectability and furthermore increase the detection window of the peptides. For most of these compounds, no or only limited pharmacokinetic data are available on human excretion. Moreover, little is known about the toxicology of the non-approved compounds and human studies are challenged by ethical considerations. Nevertheless, animal in vivo studies, in addition to in vitro simulations of metabolic reactions, have identified metabolites which represent promising targets in doping control analysis. Unfortunately, the majority of these metabolites are not commercially available.
Thus, in the current project, we plan to synthesize six proposed human metabolites of different small peptide hormones, purify and structurally characterize them by high resolution high accuracy mass spectrometry (Orbitrap FT-MS/MS; e.g. Q Exactive). Furthermore, the synthesized metabolites will be incorporated in our peptide LC-MS/MS protocol in order to proof their detectability in a single run and together with their parent compounds as well as other prohibited small peptides. Since such peptide hormones are commonly administered in low doses and additionally show extensive metabolic degradation, well-characterized reference standards of targeted metabolites are of utmost importance for a sensitive and specific detection of this group of doping agents.

Main Findings: 

In this study, eight proposed peptide metabolites were successfully synthesized employing microwave peptide synthesis, including Fmoc-based peptide synthesis methods. Subsequently, the purified peptides were characterized by high-resolution high-accuracy mass spectrometry. The peptide structures were confirmed by the accurate peptide mass (mass error ± 0 ppm - 0.7 ppm), in addition to structural information from MS-MS analysis.

Metabolites synthesized : GHRP M1, M3, M4, M5, alexamorelin M1 to M3 and leuprolide M1

Code: 15A03GS

One of the strategies for the detection of growth hormone (GH) administration is the identification of abnormal ratios between the main GH proteoforms in blood circulation. However, the high sequence similarity between them complicates a precise differentiation when relying on antibody-based quantification.

For unambiguous and precise protein identification, we will use liquid chromatography - mass spectrometry (LC-MS) analysis for the quantification of the 22 and 20 kDa GH proteoforms. Before, we will develop a sample preparation strategy for blood samples based on a novel type of (hydrogel) nanoparticles, for sample simplification and reduction of the blood matrix complexity. These nanoparticles have shown the capacity to simultaneously deplete blood from abundant proteins and enrich it in low abundance proteins through several types of particles and protocols, i.e. allowing for detection of low abundance proteins by LC-MS.

We will apply this methodology for the quantification of the 22 and 20 kDa proteoforms. We will further expand this strategy for the analysis of the 23 and 45 kDa proteoforms for which little information is available. The former showed a high concentration in post-exercise states. The latter could exist at higher amounts after administration of pharmaceutical GH. We will investigate their relevance within anti-doping analysis.

At the same time, we will purify blood extracellular microvesicles from blood samples to assess potential GH detection. These vesicles represent a blood sub-type of samples that have a much reduced matrix complexity as compared to blood. As such, the detection of low abundance proteins by LC-MS in this type of samples is significantly more accessible.

Main Findings:

Here we have explored three different strategies for GH purification from plasma samples through antibody-based, precipitation-based and ProteoCAP-based protocols, aimed for a later LC-MS/MS quantification. This quantification has also moved from the previous SRM method to a state-of-the-art PRM method that employed last-generation orbitrap-based instruments. From the three strategies, best results corresponded to precipitation based protocols that were combined with a subsequent fractionation at the protein level with disposable C4 TopTip micro-columns. Together with the PRM method and the use of trypsin, an enzyme that has a higher proteolysis efficacy than the previous Glu-C and that here we have been able to use it due to a new LC gradient configuration, our method detected GH at the spiked concentration of 5 ng/mL of each 20 and 22 kDa proteoform.

This sensitivity does not reflect the presumed true limit of detection, but it is comparable from what we obtained with our previous method, albeit here without the usage of antibodies and with potential improvements that makes
us believe that we will be able to lower these values. Currently we have three precipitation-based methods that could be all of them valid for GH detection. We have unsuccessfully tried to concatenate these methods with additional protocols for further fractionation to gain more sensitivity. We attribute these results to a too reduced protein recovery after the precipitation methods. This low recovery did maximize the impact of the protein losses that occur in any sample preparation step. This led us to escalate these protocols to provide 3-4 times higher protein recovery, where we succeed, and importantly we also succeed in removing the phospholipid contamination that was also escalated in the protocols. As a result we have protocols that now have sufficient protein amount to be amenable for concatenation of additional protocols for increased GH enrichment.

Thus, the next steps of this project will be to test the escalated protocols with and without added concatenated protocols by LC-MS to determine the assay sensitivity and to evaluate its suitability as a method for GH quantification. We believe that the strategy that we have developed in this project has the potential for detecting GH below the ng/mL frontier, thus being able to quantify GH variants at levels close to basal state.

Code: 15C17MM

Vaptans are a relatively new class of compounds with diuretic suitable for publication on WADA's website effects. They increase water excretion by inhibiting its reabsorption in the renal collecting ducts. For these reasons, vaptans have been included by the World Anti-Doping Agency in the section S5 €Diuretics and Masking agentsÅ of the list of substances and methods that are prohibited in sport. Previous investigators have reported that this class of compounds is extensively cleared by hepatic metabolism via oxidative enzymes. Consequently, the most efficacy strategy to detect them in urine samples could not be achieved by simply targeting the drug itself: the selection of one or more diagnostic metabolites is necessary. Very few data are reported in literature on the metabolic profile of these compounds in humans; at the same time, the influence of physiological and environmental factors on their excretion profile is also almost completely unexplored yet.
This project aims to define the phase I and phase II metabolic reactions and to characterize the enzymatic isoforms involved in the biotransformation pathways of vaptans. Alterations of the metabolic profile of vaptans provoked by physiological (i.e. sex, genetic polymorphism) and environmental (drug-drug interactions) factors, will be evaluated to obtain information on the impact of these factors on the biotransformation pathways detected. Finally, the possibility of obtaining sufficient amounts of vaptans metabolites by enzyme-assisted synthesis will also be explored. Human liver microsomes will be the source of the isoenzymes involved in the phase I metabolism of vaptans; the metabolites once formed will be isolated by HPLC, characterized by MS and used as reference materials to set up and validate efficacy analytical procedures to detect vaptans in urine

Main Findings:

This research project focused on the characterization of the main biotransformation pathways of vaptans with the aim to identify class-specific metabolic pathways of vaptans and select the most appropriate marker(s) of intake. 
In the first part of this project, the in vitro metabolism protocols using either pooled human liver microsomes or recombinant human CYP and UGT isoenzymes were optimized and validated in order to obtain a good correlation with the metabolism reported in humans. The optimized in vitro protocols were subsequently used to evaluate the effects of physiological (gender and genetic polymorphism) and environmental (drug-drug interactions) factors on the metabolic profile of vaptans.
Lxivaptan, mozavaptan and tolvaptan were extensively biotransformed (tolvaptan>lixivaptan>mozavaptan) mainly by CYP3A subfamily and to a lesser degree by CYP2C19 and 2D6 enzymatic isoforms to 13, 4 and 20 metabolic products respectively. Conivaptan was moderately biotransformed by CYP3A subfamily to 2 hydroxylated metabolites due to its well-known inhibitory activity against the CYP3A4 enzymatic isoform. The phase-I biotransformation pathways include hydroxylation in different positions, carboxylation, dehydrogenation, hydrogenation, N-dealkylation, isomerization and combinations of them. Most of the above-mentioned phase I metabolites once formed undergo an extensive glucuronidation by UGT2B isoforms. 
Concerning the effects of physiological an environmental factors, the results obtained showed that (i) no appreciable gender differences were registered in our experimental conditions for all the compounds under investigation; (ii) significant differences were instead registered depending on the specific allelic variant used. More in details, in the presence of the allelic variant CYP3A5 *1*1, the formation of the   phase I metabolites of second step appeared to be faster if compared to the other CYP3A5 allelic variants (CYP3A5 *3*3 and CYP3A5 *1*3) and finally (iii) the levels of all the metabolites were significantly reduced in the presence of the imidazole antifungals tested. On the contrary, in the presence of the steroidal progestins the reduction was less evident, and observed only at the highest concentrations studied.
On the basis of the above observations the most appropriate merkers of intake for conivaptan is the parent compound; the parent compound and the hydroxylated metabolites for lixivaptan; the parent compound and the de-methylated metabolite for mozavaptan; and finally the parent compound and the hydroylated and N-dealkilated hydroxylated metabolites for tolvaptan. These analytes were easily added to the LC-MS/MS analytical procedure currently used in our laboratory to detect more than 150 prohibited compounds.

Code: ISF15E01PC

Skeletal muscle size is dynamic and responsive several stimuli, among which exercise. However, while resistance exercise results in hypertrophy and increased force generation, endurance exercise leads to increased expression of the peroxisome proliferator-activated receptor (PPAR)-€ coactivator-1Å (PGC-1Å) and increased number of mitochondria, with little changes in the skeletal muscle mass. Both types of exercise stimulate the proliferation of satellite cells, muscle-specific stem cells that will subsequently differentiate and fuse with existing myofibers. Finally, exercise also increases circulating microRNAs (miRs), short noncoding RNAs that post-transcriptionally modulate gene expression. Some miRs (myomiRs: miR-1, miR-133a, miR-133b, miR-206) are abundant in the skeletal muscle and appear modulated in muscle atrophy, hypertrophy and myogenesis.
Autologous transplantation of myogenic stem cells has been proposed as the next generation doping strategy. The number of myogenic precursors may be increased both 'artificially' by autologous implantation, or physiologically, by regular exercise. However, while all myogenic precursors are increased by the implantation, exercise mainly leads to satellite cell accumulation.
The target of the study is to define if autologous myogenic stem cell implantation may effectively impinge on muscle mass and function and to clarify if circulating myomiR levels can be useful as biomarkers of myogenic stem cell abundance and type. The study will be developed by using transgenic mice hyperexpressing PGC-1Å specifically in the skeletal muscle. These mice mimic the condition of endurance-trained subjects, being characterized by high exercise performance. Briefly, circulating myomiRs will be evaluated in these mice, and correlated with the abundance and type of myogenic stem cells. Subsequently, myogenic stem cells isolated from wild-type and transgenic animals will be implanted into wild-type recipients, to test the effectiveness of the procedure in improving muscle mass/function. The results obtained with the transgenic mice will be validated on human volunteers.

Main Findings:

Skeletal muscle size is dynamic and responsive to several stimuli, among which exercise. However, while resistance exercise results in hypertrophy and increased force generation, endurance exercise leads to increased expression of the peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) and increased number of mitochondria, with little changes in the skeletal muscle mass. Both types of exercise stimulate the proliferation of satellite cells, muscle-specific stem cells, that will subsequently differentiate and fuse with existing myofibers. Finally, exercise also increases circulating microRNAs (miRs), short noncoding RNAs that post-transcriptionally modulate gene expression. Some miRs are modulated in muscle atrophy, hypertrophy and myogenesis.

Autologous transplantation of myogenic stem cells has been proposed as the next generation doping strategy. The number of myogenic precursors may be increased both 'artificially', by autologous implantation, or physiologically, by regular exercise. The target of the study has been to define if: 1)autologous myogenic stem cell implantation may effectively impinge on muscle mass and function; 2) circulating miR levels can be useful as biomarkers of myogenic stem cell abundance and type. The study has been developed by using transgenic mice hyperexpressing PGC-1α specifically in the skeletal muscle (MCK-PGC-1α). These mice are characterized by a high amount of myogenic stem cells compared to wild-type (WT) animals, and mimick the condition of endurance-trained subjects, being characterized by high exercise capacity.

The effects of the infusion of myogenic precursors isolated from WT or MCK-PGC-1α mice into Tibialis anterior (TA) muscles of WT hosts, either undergoing muscle damage or not, have been evaluated. Transplantation of myogenic precursors isolated from WT animals does not alter TA morphology and metabolism. Surprisingly, WT cell injection after muscle damage is able to restore the physiological muscle phenotype. Interestingly, when myogenic cells deriving from MCK-PGC-1α animals are infused, a shift towards the oxidative phenotype in both damaged and undamaged muscles can be observed. Histological analyses put in evidence small and oxidative fibers, while molecular markers show an increase in mRNA or protein expression levels of many markers related to myogenesis (Pax-7, Myogenin, embryonal Myosin) and mitochondria (PGC-1α, Tom20). These results suggest that the engraftment of WT myogenic precursors improve muscle regeneration after severe injury. In addition, PGC-1α overexpression might favor myogenic differentiation and affect regeneration by converting the canonical mixed phenotype of a muscle into the oxidative one, endowed with increased exercise capacity. These findings, potentially relevant to regenerative medicine aimed to improve several diseases, may also open the way to a non-ethical use as doping strategy.

As for the second aim of the study, circulating miR profile is comparable between WT and MCK-PGC-1α mice. Such result could be explained by the observation that MCK-PGC-1α mice in resting condition do not display a specific phenotype: the enhanced capacity of the skeletal muscle system, indeed, becomes evident upon stimulation, for example by exercise. Indeed, circulating levels of miR-21-5p and miR-181-5p are increased in exercised WT animals while not in the MCK-PGC-1α mice allowing to hypothesize that stress conditions up-regulate these miRs in WT animals, while the oxidative phenotype and the abundance of myogenic precursors occurring in MCK-PGC-1α mice likely improve the management of stressing stimuli. These results have been compared with those obtained in human volunteers practicing combined exercise for 3 months. Despite the small number of subjects included in the study, a trend towards increase can be envisaged for miR-27-3p, miR-133a-3p and miR-181-5p. This latter, in particular, follows the same trend in both exercised WT mice and volunteers. Despite the lack of robustness, these data suggest that circulating levels of selected miRs can be up-regulated by exercise, and that such modulation can be prevented by genetic manipulations able to improve myogenic precursor abundance and muscle oxidative phenotype. In particular, it could be proposed that low circulating levels of miR-181-5p in athletes suggest a shift of muscle metabolism from glycolitic to oxidative; this might reflect the infusion of autologous stem cells, either untreated or genetically modified. In this regard, the detection of low miR-181-5p levels in the circulation could be useful for a first screening, identifying subjects deserving more accurate analysis

Code: 15A17MT 

A variety of prohibited peptide hormones potentially enhancing athletic performance is still lacking sufficient detection methods in accredited anti-doping laboratories. This is mainly due to a yet missing adequate analytical methodology, which is crucial for detection of these very efficient (and therefore low-dosed) therapeutics and drug candidates in biological fluids (urine / blood).

Examples for new peptide-based compounds, which are explicitly mentioned on the Prohibited List, are Corticorelin (CRH) and Mechano Growth Factors (MGFs). Other peptidic compounds (e.g. Thymosin beta-4) share the same status, but are not named on the list so far although their misuse has been reported. They all are available as approved drugs (CRH) or at least subject of different clinical trials (Thymosin beta-4, MGF with different analogues).
Essential requirement for the mass spectrometric detection is the appropriate extraction of the target peptides from biological matrices to meet the required limits of detection in the low pg/mL range. This will be achieved by applying the established coated magnetic beads technology (which has been successfully applied to other peptide hormones such as insulin analogues, CJC-1295, Tesamorelin, Geref, Synacthen, etc.) with respective antibodies. The identification of isolated and enriched analytes will be realized after liquid chromatographic separation by means of high resolution mass spectrometry. Due to the unknown metabolic fate of the peptides after administration to humans, in-vitro experiments are planned, which will help to characterize amino acid sequences serving as additional analytical targets in routine doping controls. The method will be validated and the implementation into existing screening procedures (if possible) for peptides will be realized.

Main Findings: 

While the analysis of small prohibited peptides < 2 kDa is well established in doping control laboratories worldwide, the detection of peptides > 2 kDa still remains challenging due to low analyte concentrations, unstable target peptides, an unknown metabolism and a complex sample preparation. However, recent developments in analytical chemistry can help to overcome these issues and facilitate the development of mass spectrometric initial testing procedures for large peptide hormones. 

The aim of this research project was to optimize current methodologies for the detection of peptide-based drugs > 2 kDa. For that purpose, new metabolites were identified by using in-vitro experiments and the sample preparation procedure was additionally modified in order to implement further prohibited peptides. For several IGF-I and MGF analogues as well as CRH, relevant metabolites were identified, comprehensively characterized, and valid analytical procedures were developed. All metabolites comprised N- or C-terminal truncated amino acid sequences and resulted from exoproteolytic processes. In principle, the developed assays are applicable to both urine and blood (plasma) specimens, but as the renal clearance of some peptides/metabolites remains to be elucidated, blood represents the preferred biological matrix.

Code: 15B07MT 

Fc-fusion based drugs are an emerging class of pharmaceuticals which already found their way into competitive sports: EPO-Fc is an erythropoiesis-stimulating agent where the attachment of two immunoglobulin Fc domains to the protein results in a prolonged therapeutic activity due to an increased plasma half-life and enables administration via inhalation. While EPO-Fc can be simultaneously detected with other recombinant erythropoietins by routine doping control assays, there are currently no tests for other doping-relevant Fc-fusion proteins such as the myostatin inhibitor ActRIIB-Fc (ACE031) or the cytokine GDF15/Fc, which is a member of the transforming growth factor beta (TGFβ) superfamily. 
The TGFβ superfamily includes several growth factors which are promising therapeutic targets for metabolic disorders and muscular dystrophies. Both TGF-β inhibitors and the cytokines themselves have been pursued as drug candidates. Within this study, a proteomics-based detection assay for emerging Fc-fusion proteins relevant as performance-enhancing agents in sports will be developed. ActRIIB-Fc (ACE-031), a fusion protein composed of the extracellular domain of the human activin receptor type IIB (ActRIIB) and the Fc domain of human immunoglobulin G (IgG), which was found to significantly increase muscle mass and function by inhibition of myostatin and other ligands of ActRIIB, will be used as model compound in addition to GDF15/Fc, a fusion protein of the TGFβ cytokine growth/differentiation factor 15 (GDF15) and the immunoglobulin Fc fragment. Different peptides, mutants and constructs of GDF15 are currently tested as pharmaceuticals for the treatment of different age-related and metabolic disorders. 
The proactive development detection assays for therapeutic Fc-fusion proteins, TGFβ cytokines and TGFβ inhibitors is of great interest as several drugs of these categories are already available on the black market as well as for research purposes. 

Main Findings: 

Cytokines of the TGF-β superfamily as well as their inhibitors are currently being evaluated as potential protein drugs for the therapy of a variety of diseases such as metabolic disorders and muscular dystrophies. The aim of this research project was to develop detection assays for two emerging Fc-fusion proteins potentially relevant as performance-enhancing agents in sports: The TGF-β cytokine GDF-15/Fc and an ActRIIB-Fc fusion protein related to the TGF-β/myostatin inhibitor ACE-031. Two complementary, multiplexed detection assays for ActRIIB-Fc were developed by using affinity purification in combination with either proteolytic digestion and LC-HRMS, or Western blotting. Both approaches can readily be modified to include other ActRII-Fc fusion proteins such as Luspatercept (modified ActRIIB-Fc). A central and critical aspect of the test methods is the ability of all target analytes to bind to activing-receptor ligands; in case of the model compound GDF-15/Fc, the available reference materials were found to be of substandard quality and w provided as multimer, which significantly interfered with affinity purification process due to the elimination of its binding capability.  Consequently, this analyte was not included into the test method validation process but the analyte’s characteristics and its potential implementation into flexible and expandable assays was elucidated. Overall, the developed assays proved fit-for-purpose and will expand the range of available test methods for emerging protein therapeutics, particularly those of higher molecular mass.