Projets de recherche

Code: 16E05AB 

Since 2003, gene doping has been included in WADA’s list of banned substances and methods. Extensive research by us and other investigators has led to the development of a method for gene doping detection that directly targets a doping gene using highly-specific and sensitive PCR assays. The efforts of our laboratory led to the development of a test for erythropoietin (EPO) gene doping and its recent implementation in the Australian Sports Drug Testing Laboratory (ASDTL). Based on the successful implementation of the test at ASDTL and in response to WADA’s recommendations, we now propose to evaluate approaches to further improve the test by increasing its sensitivity, reducing its cost and streamlining the test protocol.  
The outcomes of this project will improve the EPO gene doping test. Importantly, this work will allow the first gene doping test to be integrated into the arsenal currently used in doping control and to bolster the fight against doping in sport. 

Main Findings: 

As a result of this research, we propose several modifications to the EPO gene doping test that will enhance its sensitivity and make it cheaper and easier, while maintaining reliability. These include an alternative method to identify PCR false positives and a method to concentrate the genetic material extracted from samples prior to analysis. We have extended the test from doping material in plasma to doping genes that might be associated with blood cells and have demonstrated that blood samples kept frozen at -80°C for at least three months are suitable for testing. This broadened scope increases the utility of the test for gene doping detection. We found that extracting genetic material from whole blood rather than its fractions provides operational robustness for gene doping testing. These improvements to the test will facilitate its implementation and use in doping control.

Code: ISF16D02NL

Proteins involved in erythropoiesis and iron metabolism have been demonstrated as potential biomarkers to detect blood doping. Hepcidin, a peptide hormone that is a regulator of iron homeostasis, is reported to be regulated by recombinant human erythropoietin (rhEPO) administration and by the autologous blood transfusion. It was suggested that measurement of iron in ethylenediaminetetraacetic acid (EDTA) plasma may be a costeffective marker for the screening of blood transfusion use. Increased levels of EDTA plasma iron were also detectable using a fast automated method. Thus, EDTA plasma iron may provide further evidence of blood manipulation. In 2014, erythroferrone (ERFE) was identified as a novel erythroid regulator of iron metabolism in a mouse model. These observations suggest that ERFE may be a potential biomarker in the detection of blood doping. However, the ironomics strategy to detect blood doping could be impacted by iron injection performed by athletes and be considerated as an confounding factor for iron metabolism biomarkers. In this project a clinical study will be performed to investigate the potential confounding effect of iron injection on iron metabolism biomarkers.

Main Findings:

Proteins involved in iron metabolism, such as hepcidin, are potential blood doping biomarkers. Monitoring of these markers could offer a strategy to complement the actual longitudinal follow-up of hematological parameters measurement, such as reticulocyte percentage (Ret%), to detect blood doping. Iron injections, which are frequently supplied to athletes, could affect hepcidin concentration and therefore, present a confounding factor in hematological profiling for blood doping detection. In this project, urinary iron was tested as a novel biomarker to monitor iron injection. 

A randomized, single-blind, placebo-controlled trial was conducted in male volunteers who received a single intravenous injection of ferric carboxymaltose or placebo. The effects of iron injection on iron metabolism markers and hematological were investigated. Hepcidin concentration was measured in blood by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) and the urinary excretion kinetics of iron was quantified by inductively coupled plasma mass spectrometry (ICP-MS).

Intravenous iron supplementation increased Ret% significantly, and also serum hepcidin concentration was increased 16-fold relative to baseline. Interestingly, urinary iron concentration was increased by 12-fold at 3 h after iron injection, and it remains significantly elevated until day 1 after administration. A specificity of 100% and a sensitivity of 79.3% were achieved with a proposed threshold of 251 ng/mL for urinary iron.

Due to the impact on Ret%, iron injection is a cofounding factor in evaluation of the hematological parameters for the detection of blood doping. Furthermore, urinary iron quantification could offer a novel strategy to monitor intravenous iron injection in the doping control samples.

Code: 16A06MM 

When athletes dope the drugs are changed by the body and excreted in the urine. These drug metabolites must be processed by anti-doping laboratories to enable detection using a range of sophisticated techniques. An enzyme called beta-glucuronidase, isolated from Escherichia coli bacteria, is routinely used by anti-doping labs to process samples prior to analysis. It has become an essential tool used by analysts in the fight against doping.  Unfortunately, this beta-glucuronidase enzyme only works on some drug metabolites called glucuronides leaving others called sulfates unprocessed, and so doping may go undetected. Creating a mild and universal enzyme to process sulfate metabolites would significantly improve anti-doping analysis. 
In earlier WADA-funded research we engineered an enzyme from the bacterium Pseudomonas aeruginosa called an arylsulfatase that is able to process the sulfate metabolites that E. coli beta-glucuronidase cannot. Our work improved enzyme activity for testosterone sulfate hydrolysis by over 270-fold and increased the substrate scope. However, the activity for some drug metabolites remained low leading to inefficient hydrolysis. In this project will employ laboratory-based methods of rapid evolution to enhance the substrate scope of the P. aeruginosa arylsulfatase enzyme for anti-doping applications.
The project outcome will be mild and universal arylsulfatase enzymes for processing drug metabolites that will complement E. coli beta-glucuronidase. The new enzyme will be rigorously evaluated by the WADA-accredited Australian Sports Drug Testing Laboratory. Including the improved enzyme in the methods used to process drug metabolites will increase the sensitivity of analysis and allow doping to be detected for a longer period after an athlete takes a banned drug. We expect this improved P. aeruginosa arylsulfatase will join E. coli beta-glucuronidase and also become an indispensable tool used by anti-doping laboratories in the fight against doping.

Main Findings: 

Steroid abuse still makes up a large proportion of the incidences of doping in world sport. This abuse leaves tell-tale metabolites in the urine. To date, anti-doping labs have focussed on one class of steroid metabolites: those with glucuronide conjugates. There is now a wealth of evidence suggesting that another class, steroids with sulfate conjugates can in some cases provide longer lasting markers of doping. However, steroid sulfates are difficult to detect by gas chromatography-mass spectrometry (GC-MS) methods that are essential for producing evidence in suspected cases of doping.
Before GC-MS analysis, steroid metabolites must be prepared by first hydrolysing the glucuronide or sulfate conjugates. Most glucuronides can be efficiently hydrolysed by a bacterial enzyme, but no general sulfatase enzyme is available to hydrolyse the sulfate esters. The aim of this WADA-funded project was to engineer a sulfatase enzyme to meet this need.
WADA’s first grant for sulfatase engineering (WADA 13A13MM) allowed us to find and optimise mutations in Pseudomonas aeruginosa sulfatase (PaS). The best combinations of mutation allowed PaS to hydrolyse testosterone sulfate (TS) 150 times faster than the original bacterial enzyme. However, this version of PaS, like its predecessors was biased towards steroids with a beta configured hydroxyl group, such as TS, or dehydroepiandrosterone sulfate (DHEAS). The alpha configured steroid sulfates such as epitestosterone sulfate (ETS), androsterone sulfate (AS) or etiocholanolone sulfate (ECS) were hydrolysed thousands of times slower, if at all.
This one year WADA follow-up grant (WADA 16A06MM) has allowed us to take one PaS variant (named PVFV-PaS) that had significant ECS activity and engineer it towards the alpha configured steroid sulfates. We used genetic engineering to prepare thousands of PaS genes with mutations scattered in regions that we had discovered to be important for binding steroid sulfates. Our assays examined thousands of these variants in microlitre-scale reactions to find the best mutations for ECS hydrolysis. The work resulted in the identification of several new beneficial mutations: the best combination resulted in 15 times more activity towards ECS compared with PVFV-PaS. Over two WADA-funded projects, we have taken an enzyme with no detectable activity for alpha configured steroid sulfates and have prepared a variant with enough activity to be applied in anti-doping laboratories. Our work has also developed the know-how to prepare gram quantities of this purified enzyme from two litres of bacterial culture: enough to process more than a litre of urine samples for GC-based analysis.
The project also tested several PaS variants with pooled urine samples to evaluate which endogenous steroids could be detected when compared with no treatment or a typical beta-glucuronidase treatment. The GC-mass spectrometry method detected 38 steroids, 14 of which were enhanced by PaS treatment. That is, without PaS treatment, 14 steroid sulfates did not contribute to the GC-MS steroid profile. Further analysis revealed that eight steroid signals were enhanced by PaS treatment compared with the industry standard beta-glucuronidase and five were enhanced by using an engineered PaS variant compared with the original PaS enzyme. 
In conclusion, we have developed sulfatases that can hydrolyse many of the steroid sulfates important for anti-doping analysis under similar conditions as already used for steroid glucuronide hydrolysis. The studies have revealed fundamental knowledge about the sulfatase enzymes (ACS Catal. 2018, 8, 8902−8914) and have found application in sample preparation prior to anti-doping analysis (Drug Test. Analysis 2017, 9, 1695-1703; Analytica Chimica Acta 2018, 1030, 105-114).

Code: 16A05MT

Corticoids are prohibited in elite sport for in-competition testing and when systemically applied (oral, i.v., i.m. etc.). In contrast, local/topical administration is permitted. For urine analysis the technical document (TD2014 MRPL) recommends a MRPL of 30 ng/mL and levels below the MRPL should be reported as negative.

There are no specifications about concentrations of corticoids in blood, although blood levels directly correlate with the route of administration. Hence, quantification of synthetic corticoids in blood can provide information whether an athlete was under the influence of systemic corticosteroid influence at the time of competition or not; largely independent from the route of administration. The quantification can be performed from a drop of dried blood (dried blood spot, DBS), which can readily be taken from an athlete in addition to a urine specimen. Sampling, transport and storage of DBS is easy and the analysis is nearly completely automatable. The obtained results provide the desirable information about the amount of the active, circulating corticoid just before or shortly after the competition. This will enable a superior basis to decide whether the detected amount of the drug was of benefit to the athlete or not without causing expensive or invasive additional sampling needs.

Main Findings:

Synthetic glucocorticoids belong to the classes of substances that are prohibited in-competition only and for which permissible as well as prohibited routes of administration exist. Consequently, attributing findings of corticoids in doping control samples to time-points and routes of administrations has been of particular importance but, at the same time, a considerably challenging task. In order to complement information obtained from urine analyses, the utility of dried blood spots (DBS) was assessed and a quantitative method was established allowing to determine whole blood concentrations of synthetic glucocorticoids for sports drug testing purposes. The assay was fully validated and yielded figures of merit enabling the quantification of glucocorticoids at pharmacologically relevant concentrations as demonstrated with single-dose proof-of-concept elimination studies. Dexamethasone, methylprednisolone, and prednisone (plus its metabolite prednisolone) were determined in post-administration DBS samples for 9-24 h and observed concentrations reached, depending on the administered drug, values of up to ca. 200 ng/mL. The analytical approach employed an automated DBS extraction utilizing stable isotope-labeled dexamethasone as internal standard and subsequent liquid chromatographic-mass spectrometric detection. Additionally, stability studies were conducted over a period of 90 days in order to assess the overall suitability of DBS as alternative matrix for sports drug testing. All model compounds were found to be stable over the entire storage time independent from the parameters light, humidity, and storage atmosphere.

In consideration of the obtained results, a strategy and concept of complementary DBS sampling and testing evolved, which can significantly contribute to addressing the aforementioned challenges concerning compounds prohibited in-competition only. Given the minimal-invasive nature of DBS combined with its low-cost sampling and storage requirements, DBS can be considered as additional test matrix collected in concert with any doping control urine sample taken from athletes in-competition. In case of urinary glucocorticoid concentrations exceeding the reporting threshold of 30 ng/mL, the concurrently collected DBS sample can be analyzed to provide additional evidence concerning the presence (or absence) of pharmacologically relevant blood levels of the glucocorticoid. Depending on the data obtained from DBS analyses, i.e. if (according to blood concentrations) the athlete was under the influence of glucocorticoids at the time of competition, the decision-making process at the result management level is facilitated. The strategy can further be expanded to other banned substances such as stimulants (e.g. cocaine, amphetamine, etc.) as well as narcotics.

Code: ISF16M04JP 

There is evidence that changes occur in red blood cells (RBCs) stored ex-vivo (e.g. in a blood bag or as a frozen concentrate) that do not occur in a normal RBC population. The underlying research proposal follows-up the empirical selection of recombinant antibodies able to selective recognize blood samples having been stored using regular procedures (i.e. bags of RBC concentrates kept at 4ºC or frozen) already performed as part of a first year granted project (HemAb). Our team successfully selected several clones from a large phage display antibody library that selectively recognize stored blood. 
To complete the Proof of Concept phase of the project we propose to do as follows: 
1.- Optimization of the selected clones 
Especially we will increase the valency of the recombinant antibodies from the current monovalent stated. In addition we will examine various tag-sequences to identify the optimal tag, considering the final detection system and last we will optimize the affinity of the most ideal recombinant antibody if needed. 
2.- Testing of the different clones in Flow Cytometry conditions with blood samples under different storage conditions. - Fresh blood samples
- Blood samples stored at 4ºC > 30 days (including kinetic samples taken along the storage period)
- Blood samples stored frozen >30 days
3.- Optimization of the FC methodology
Fluorophores, combination of antibodies, pre-cell enrichment, etc.
4.- Development and validation of the scale-up production of the selected clones.  To ensure that the assay can be transferred to partner laboratories large scale production of the most optimal recombinant antibody will be optimized and a production strain will be frozen.
5.- Testing of real samples of transfused individuals.
- Blood samples from transfused patients (homologous, HBT)
- Blood samples from transfused volunteers (autologous, ABT)

Main Findings: 

AIMS
The main objective of the project was the development of recombinant monoclonal antibodies able to recognize changes in red blood cells (RBCs) specifically related to storage conditions (blood bags). The antibodies will be used to develop a method to unambiguously identify the use of blood transfusion, either autologous (ABT) or homologous (HBT). This is a follow-up project.

RESULTS
Using Phage Display, a series of 133 different clones were selected showing some selectivity towards recognizing stored RCs with respect to freshly collected RBCs.

Two different selection procedures and two different testing procedures were assayed along the selection procedures, i.e. ELISA and/or Flow Cytometry.

After testing those selected clones under different conditions, trying to expose and recognise the RBC storage-specific antigens, both surface or cytoplasmic, one of them was able to selectively detect stored RBCs in the presence of a vast excess of fresh RBCs.

CONCLUSIONS
Despite initial results showing the selection of some promising clones able to selectively differentiate stored RBCs from fresh RBCs, none of the clones worked properly under Flow Cytometry conditions in any experimental set-up tested. New rounds of selection were made without fruitful results

Code: R16R01WS 

In this research project urinary excretion profiles and corresponding urinary detection windows of Mildronate (Meldonium) after single and multiple oral applications of the drug should be investigated in healthy volunteers. The results of the study will have an immediate impact on current Adverse Analytical Findings of Mildronate. The obtained pharmacokinetic data will help to interpret the estimated urinary concentrations and may be helpful in cases, where athletes claim that they have taken high doses of Mildronate before the compound was implemented in the 2016 WADA prohibited list.

Within the research project the following activities are planned: 1. Application for ethical approval

2. Administration studies after single- and multiple oral applications of Mildronate

(dosages and interval of drug ingestion based on recent publications and clinical recommendations [8,9]).

o single-dose phase: volunteers receive one dose of 1500 mg Mildronate and collect all urine samples for four to five consecutive days.

o multiple-dose phase: volunteers receive 500 mg Mildronate three times a day for six days. During this administration period the volunteers should collect all urine samples. After the application period (from day 7 on) the volunteers should collect one urine sample per day until the urine is blank.

o The volunteers should provide information about their anthropometric data (age, height, weight) and the date and time urine samples were collected.

3. Analyzing of urine samples using LC-MS/MS and HILIC/HRMS/MS approaches. [4]

4. Investigation of urinary excretion profiles of Mildronate and a comparison of the urinary detection windows after single and multiple oral applications of the drug.

5. Publication of the data in an international journal

Main Findings: 

Preliminary results: So far, only one excretion study is published, which shows the urinary detection window for the application of a single dose of 250 mg Mildronate. In this study Mildronate was detectable up to 50 hours after intake. [7] However, the pharmacological dosage recommendation for Mildronate is several times higher.

Furthermore, two studies demonstrate pharmacokinetic profiles of Mildronate: 1.) after intravenously administration and 2.) after oral application in human plasma. These findings suggest, that multiple administration leads to accumulation of Mildronate in human plasma. [8,9] No data are available concerning the excretion profile after single and high doses of oral administered Mildronate in urine.

Ce document n'est actuellement disponible qu'en anglais.

Code: R15M02RC

This project will assist in establishing an analytical laboratory for testing nutritional supplements to detect prohibited substances such as steroids, stimulants and other performance-enhancing drugs. The project will provide the necessary financial assistance to fund a full time instrument technician for a period of one year and a contribution towards the cost of method validation and laboratory accreditation. The funding provided by this project will supplement a substantial, ongoing (LIVE) investment by the University of Hertfordshire to acquire state-of-the-art analytical instrumentation.

Main findings

A simple and straightforward approach for determining MRPL for the detection and identification of prohibited substances in dietary supplements has been followed. A keyword search for recommended doses of prohibited substances was conducted, followed by the collection of recommended daily intake of a range of dietary supplements. These data were then used to calculate MRPL from which the limit of detection (LOD) for each prohibited substance was derived. The corresponding MRPL-derived LOD were found to be three orders of magnitude greater than those determined experimentally from the in-house, multiplex assay. From these data, it has been possible to estimate an LOD that would be anticipated as an absolute maximum for each substance intended to be used as part of our validation [9]. It also facilitates the considerations and parameters necessary for the reporting of findings from any future quantitative methods that will be developed. Currently, substances administered via routes other than oral have been included; further considerations are required to determine whether there is potential for their inclusion in solid dietary supplements before eliminating them.

Code: 15C13MZ 

According to WADA, the issue of beta-2 agonists will continue to be a focus of its research activity in order to ensure that the administration of large doses of these substances is prevented and prohibited, but the appropriate care and treatment of asthmatic athletes is facilitated. In this project we will conduct a human pharmacological study to investigate dosing and combinatory beta-2 agonist effects. Single vs. combinatory threshold doses of non-prohibited, short-acting (salbutamol) and long-acting (formoterol) beta-2 agonists will be administered by inhalation and potential additive effects will be investigated by measuring skeletal muscle metabolic and hypertrophic signaling, endurance performance (10 min cycling time trial) and cardiopulmonary function (cardiac output and VO2max). The main objective of the proposed study is to investigate dose-dependent additive/synergistic effects of short- and long-acting beta-2 agonists in terms of skeletal muscle metabolism/hypertrophy, endocrine regulation, cardiopulmonary function and endurance performance.  

Main Findings: 

Results: All medication combinations were reliably detected in the urine samples by LC-MS/MS meeting WADA standards. None of the samples collected after application of verum medication resulted in concentrations exceeding the threshold concentrations set for doping control analysis. Mean Power Output during TT was not different between the different study arms. There was a treatment effect regarding lung function observable without any influence on performance or health. There was a treatment effect on myocardial contractility measured by Echocardiographic Longitudinal Strain which increased for both strains and there was a marked effect of combined treatment. 

Microarray subsample analysis revealed no significant treatment effect on gene expression of NR4A1 or NR4A3, but an effect was observable for NR4A2 with the most significant difference between Placebo and salbutamol+formoterol. The β2-combination influenced up- and downregulation of differently expressed genes most compared to the other study arms. Muscle analysis did not show any treatment effect on NR4A protein and NR4A1/NR4A3 gene expression, whereas a whole group treatment effect was observable for NR4A2. Further pathway analysis with gene expression software TACx and linked WikiPathways revealed treatment effects in energy metabolism related genes ATF3 (e.g. Hypertrophy model; TGF-beta signaling pathway), PDK4 (e.g. Estrogen receptor pathway; nuclear receptors meta-pathway), LPL (e.g. Metabolic pathway of LDL, HDL and TG; PPAR signaling pathway), CREM (e.g. mBDNF and proBDNF regulation of GABA neurotransmission), and ATP1B3/ATPase (e.g Calcium regulation in cardiac cells).

Noradrenaline, adrenaline and TGF-β concentrations in blood were not affected by treatment or gender, whereas IGF concentrations showed a treatment effect 24h Post compared to Pre for women. CO data determined by Clearsight® device were not reliably reproduced in all measurements due to technical artefacts. 

Both β2-agonists stimulated hypertrophy in a dose dependent manner compared to negative control in C2C12 myotubes. Diameters relative to control were increased for all β2-agonists treatments, but an additive effect were clearly observed for salbutamol+formoterol compared to control or the respective β2-agonists alone.

Discussion: There is presumably no performance enhancing effect in this study design with the used doses of β2-agonists either alone (salbutamol or formoterol) or in combination (salbutamol+formoterol) compared to Placebo, whereas it was shown that in cell culture, β2-agonists may indeed have a strong hypertrophic effect and exert their effects in an additive manner that can be relevant for human in vivo pharmacologic kinetics.

An acute effect on the lung function was observable without side effects and with presumably no impact on exercise performance capacity in healthy subjects. Acute effects were observable for heart contractility but without objective impact on aerobic performance capacity or health.The impact of chronic β2-application in healthy and asthmatic subjects on TT performance of 
longer duration, which simulates real life competition even closer, has still to be determined.

Code: ISF15E10YP 

The current research on the molecular signature of rHuEPO doping has, so far, provided some evidence that “omics” technologies such as transcriptomics have the potential to significantly strengthen the current ABP approach and contribute to other traditional anti-doping tests. This approach if successful can in the future be applied to the detection of other doping substances and methods difficult to detect such a recombinant human growth hormone and blood transfusions. There is also the interesting possibility that an "omics"-based approach could help reduce the pressure on the anti-doping obligations of athletes such as the “athletes whereabouts”. In order to confirm that an integrative “omics” approach is a possible solution to improve rHuEPO detection, it is of paramount importance to precisely determine normal gene expression reference values as well as to carefully assess the potential effects of external factors on blood gene expression profiles, such as prior training, altitude including different hypoxic “dose” and protocols, sport discipline, level of competition, gender, ethnicity and age. The investigations proposed are necessary before including the promising blood gene biomarkers in the ABP and/or the development of a stand alone test to reveal doping or identify suspicious samples for targeting purposes.