Projets de recherche

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.

Code: 15A31JP 

A project (acronym GOpep) was approved by WADA with the objective of detecting a Neu5Gc containing EPO O-glycopeptide using latest generation MS instruments (i.e. AB Sciex Qtrap 6500).

The EPO O-glycopeptide shows the lowest glycan heterogeneity, thus being the best choice to reach the necessary MS sensitivity. Results obtained showed that the glycopeptide isoform containing Neu5AcNeu5Gc was found to be the most abundant form with its triply charge species at m/z 810.3 giving the best signal. A limit of detection of around 2 IU EPO/L, from a standard preparation was achieved, compatible with the expected concentrations in human urine. An antibody against the peptide was also developed and initial results show that it also recognizes the glycopeptide. However, matrix effect when spiking real samples at very low concentrations,
as well instrumental conditions to speed-up the analysis are still to be solved.
The hypothesis of this project is that MS sensitivity has reached a status in which EPO glycopeptides, particularly O-glycopeptides as they present lower heterogeneity, are detectable in urine or blood samples. Using a proper combination of specific peptide immunopurification with other desalting techniques, matrix effects can be avoided and the required sensitivity for the EPO O-glycopeptide containing the non-human tag (Neu5Gc) reached.

Objectives:
1.- To improve sample clean-up by using the already developed polyclonal antibody against the peptide backbone and other desalting techniques.
2.-. To improve the nanoLC-MS/MS set-up using monolithic columns for high throughput and on-line sample clean-up.
3.- To develop monoclonal antibody for future use of the methodology if the use of the polyclonal proves successful.
4.- To validate the procedure in urine and serum samples

Main Findings:

The main objective of the project was to develop an MS-based analytical procedure for the detection of an EPO O-glycopeptide containing the non-human monosaccharide N-glycolyl-neuraminic acid (Neu5Gc) as an unambiguous proof of the exogenous origin of the hormone (i.e. rEPO or analogues). The trypsin released EPO O-glycopeptide T13: (E117-R131) shows the lowest glycan heterogeneity, thus maximizing signal sensitivity while the peptide backbone will make it unique for EPO.
Through the method development, it was found that the formation of ubiquitous ammonium adducts was unavoidable, even under conditions where no ammonium salts were used.

The ammonium adducts of the doubly and triply charged species of the “endogenous” species (T13 O-2Neu5Ac) have masses identical (within 0.5 m/z) to the non-adduct forms of the “exogenous” species (T13 O-1Neu5Ac-1Neu5Gc) containing a 13C atom. The potential risk of having false positives forced the development of a method in which there was a complete chromatographic separation between these two very similar species.

The overall results demonstrated that the “exogenous” glycopeptide (T13 O-1Neu5Ac-1Neu5Gc) could be detected in rEPO using both a QTRAP6500 (low resolution) and an Orbitrap Fusion Lumos (high resolution). This approach includes the development of an MRM and PRM method respectively and the enrichment of the target EPO T13 O-1Neu5Ac1Neu1Gc using the anti-EPO T13 antibodies.

The method developed in this project is relatively straightforward, however the LOD using the most sensitive instrument in the market is still far from its applicability for real biological samples.

Still, as opposed to SAR-PAGE method, the proposed strategy may have the potential to unequivocally identify rEPO by detecting the 1-2% of T13 O-1NeuAc1NeuGc present in the sample using the signal of T13 O2Neu5Ac, which is present in all forms of EPO (exogenous and endogenous), as its own internal standard and quality control. The peptide chosen (T13) is unique for EPO, so it might allow the development of specific immunopurification techniques.

Code: ISF15D14ZG

The ability to increase oxygen carrying capacity to exercising skeletal muscles is a highly effective method for improving athletic performance. Unfortunately, some athletes seeking to gain an edge over their competition, have turned to artificially enhanced performance gains through blood transfusions, despite these methods being banned by the World Anti-Doping Agency. While methods such as flow cytometry can reveal heterogeneity in red blood cell (RBC) surface antigens and thereby detect homologous transfusions – or blood doping from a different person, there is currently no method available to detect autologous blood transfusions (ABT) with an athletes own blood. The lack of a direct detection method represents a significant problem for endurance sports, and the absence of a test means that this performance enhancing method is still widely utilized. There is evidence that biochemical changes occur in RBCs stored ex-vivo, including changes in their cell membrane that do not occur in a normal RBC population. One major obstacle to the development of a specific and reliable method for detecting ABT is then the lack of an available technique for detecting these age-related changes in circulation and at low concentration.  
The goal of this proposal is to develop the ability to quantify these modifications in red blood cell storage age using a combination of dielectrophoretic spectroscopy and a storage sensitive membrane cross-linking reaction, and to employ this approach to develop a simple and specific test for the detection of autologous blood transfusions in endurance athletes. The successful outcome of this project will lead to the development of an entirely new electrical approach to monitoring an athlete’s blood sample, and will lead to a new ABT indicator that is simple, rapid, require only a small droplet of blood, and capable of being integrated into an athlete's Biological Passport. 

Main Findings:

Background. The ability to increase oxygen carrying capacity to exercising skeletal muscles is an effective method for improving athletic performance. Unfortunately, some athletes have turned to artificially enhanced performance gains using blood transfusions, despite these methods being banned by the World Anti-Doping Agency (WADA). One main limitation in detecting autologous blood transfusions (ABT) is that there is no direct method capable of performing specific detection across a large transfusion regime, including detecting re-infusion with a small volume of blood under conditions when re-infusion has occurred multiple weeks prior to a competition. The significance of this project is based on this effort to develop a new method to overcome this problem using a combination of electrokinetics and microfluidics.

Results. We used electrodes to measure the electrical behavior of RBCs. To perform our experiments, RBCs were collected from healthy human volunteers and stored in storage buffer. We discovered that the electrical properties of RBCs change when cells are stored. We also developed an ABT assay that can quantify differences in mechanical elasticity of RBCs based on the ability for RBCs to deform in a microfluidic channel.

Conclusions. We believe that these two ABT assays complement each other and speculate that the microfluidic deformability assay can be used as a rapid screen for athletes to detect potential doped subpopulations of RBCs. The electrokinetic assay could then be used as a secondary detection method to verify the presence of aged RBCs. We are looking forward to evaluating the performance of these assays on in future work from samples collected from doping volunteers.