Projets de recherche

Code: 18C08MH

Salbutamol is a short-acting beta2-agonist used for asthma and exercise induced bronchoconstriction and is permitted in inhaled doses up to 1600 μg in 24 h, not exceeding 800 μg in 12 h. A corresponding urine threshold of 1000 ng/mL and decision limit of 1200 ng/mL has been established to discriminate permitted inhalation from prohibited misuse. If an athlete exceeds the decision limit in doping control, this is recorded as an Adverse Analytical Finding (AAF) that may result in the athlete being charged with an anti-doping violation.

While salbutamol has been available for half a century, knowledge about its pharmacokinetics in relation to athletes and anti-doping control is incomplete. A handful of clinical trials underpin that high urine concentrations of salbutamol, relative to the dose inhaled, can occur, indicating that the risk of AAFs reported for salbutamol is possibly greater than previously assumed. These observations are further substantiated by the fact that several supplements and medications may interfere with the metabolism and subsequent excretion of salbutamol, which to our knowledge, are unexplored with respect to the salbutamol decision limit. Thus, athletes are exposed to several factors that may affect the pharmacokinetics of inhaled salbutamol, including, but not limited to strenuous exercise, dehydration, diet and supplements, and other medication.

This project will provide a comprehensive investigation into the factors that affect the intra- and inter-individual pharmacokinetics of salbutamol in female and male athletes. The primary objective is to model the peak urine excretion rate and peak urine concentrations of unchanged salbutamol and its major 4'O sulfate metabolite after permitted inhalation and prohibited oral ingestion to possibly improve the predictive value and accuracy of the decision limit for salbutamol.

Main Findings

The 2021 Prohibited List allows athletes to administer salbutamol at inhaled doses up to 1600 µg in 24 hours, not exceeding 800 µg in 12 hours. A urine threshold of 1000 ng/mL with a corresponding decision limit of 1200 ng/mL has been established to discriminate permitted therapeutic use from prohibited supratherapeutic use during doping control. Despite many studies investigating the pharmacokinetics of salbutamol, limited data are available on factors that may confound its pharmacokinetics. In this project, we investigated the effect of dietary flavonoid intake, dehydration, combined flavonoid intake and dehydration, and consecutive days of salbutamol inhalation on urine pharmacokinetics in highly trained females and males who were subjected to prolonged strenuous exercise. Additionally, we evaluated the diagnostic performance of the current threshold and decision limit in discriminating permitted inhaled doses (1600 µg in 24 hours) from prohibited oral doses of 8 mg salbutamol. In this comprehensive repeated measures study, we collected and analyzed more than 750 urine samples and demonstrated that the current decision limit approach, whereby concentrated urine samples are adjusted to a specific gravity (SG) of 1.020, has robust sensitivity and specificity for application in doping control. Only 1 sample, collected from a female participant following consecutive days of inhalation, exceeded the decision limit after SG-adjustment (reaching 1303 ng/mL), and only 4 samples (from 3 different participants; also all from consecutive days of inhalation) exceeded the threshold with SG adjustment. As such, employing SG-adjusted data from 0-6 h at all trials, which is the most relevant window of detection, sensitivity at the decision limit was 40% with 99% specificity, while at the threshold, sensitivity was 47% with 98% specificity. Females generally presented with higher urine concentrations of salbutamol than males, and 3 of the 4 samples exceeding the threshold were from female participants. Consecutive days of inhalation resulted in higher urine concentrations of salbutamol, indicating that 24 hours are insufficient for complete urine 2 elimination of salbutamol for most individuals. Prior bioflavonoid intake lowered urine concentrations of salbutamol after inhalation, while dehydration, as expected, led to higher urine concentrations of salbutamol which could be accounted for with SG adjustment. In conclusion, the current threshold approach with SG adjustment displays robust sensitivity and specificity, even in the face of dehydration, bioflavonoid intake, and consecutive days of treatment. Female athletes generally present with higher urine concentrations than male athletes.

Code: 18D06XD 

Among the substances declared by the athletes during the sample collection sessions and reported in the doping control forms (DCF), it is quite frequent to observe supplements or medication based on thyroid hormones. This supplements or medications contain not only levothyroxine (T4) or triiodotironine (T3) but some of their derivatives as Triacana (3,5,3’-triiodothyroacetic acid) or Tetrac (3,5,3’,5’-tetraidothyroacetic acid). In 2017, at the WADA accredited antidoping laboratory of Rome, the athletes declaring to consume this kind of substances, was ten times higher compared to the prevalence of hypothyroidism in Italy. This high incidence of their consumption among the athletes in conjunction with their metabolic actions and the consequences of their intake over the health, impose to investigate which is the real use of these compounds and to start to investigate a new potential doping practice.

We plan to investigate the real analytical possibilities for determining the prevalence of use in sports using the available capabilities in the antidoping laboratories, ideally in already existing methods starting from urine or serum samples collected for other antidoping analyses.  This would allow, if considered convenient, to include an additional section in the athletes biological passport (ABP) endocrinological module for these hormones.

The main goal is to monitor thyroxine hormones (TSH, freeT3, freeT4 and freeT4/freeT3 ratio) in athlete’s serum and investigate the best biomarkers in urine focusing the attention in their potential inclusion in the ABP endocrinological module. Once the method developed and biomarkers chosen, the proposed approach will be applied to different thyroid conditions and the profiles under some administrations evaluated. 

Main findings

A method to detect thyroid hormones (TH) and some related metabolites in serum was validated using the LCMS/MS technique. The correlation of the validated method with RIA showed adequate results and one of the advantages of the LCMS technique with respect to RIA is that it does not show cross-interferences as is the case of T3 with triiodo-thyronacetic acid, which could happen with other metabolites. One of the disadvantages is the matrix effect, so it must be treated with particular care in the implementation process. Analysis of serum samples from athletes who reported levothyroxine consumption showed elevated levels of thyronine and tetraiodo-thyronacetic acid. At least preliminarily, these two compounds and the relationships that involve them (with T3 and T4) could be used as markers for the detection of levothyroxine consumption in serum.

Also, a method was validated for urine that allows the detection of THs and their metabolites. The method was applied to the total urine fraction (free + glucuronide conjugates + sulfate conjugates) but can be applied to the fractions separately. It was based on extraction at neutral pH to have a global idea of the excretion in urine of acidic (thyronacetic acids) and basic (thyronamines) metabolites.

Applying the validated method in LCMS to the total fraction of serum and urine, it was possible to verify that the levels of T3 and T4 of athletes are lower than those of a euthyroid population even when they are under the administration of TH supplements, coinciding with episodes of hypothyroidism described in reports focused

on high-performance sports. However, the high efficiency of the HTP axis (which proved to be, throughout the study, the most strict, refined, and exquisite) does not allow us to observe differences between athletes who do not declare and those who declare having used TH supplementation.

Direct measurements of T3 and T4 do not seem to be useful in these cases since everything seems to indicate that external supplementation aims to cover the needs of thyroid hormones to maintain homeostasis. Therefore, it could be assumed that the metabolic pathways observed in a euthyroid individual after the administration of TH are not the same as those observed in athletes. The detection of the administration of TH, at least triiodothyronine (T3) and levothyroxine (T4) in urine could work when dealing with euthyroid individuals, either by applying cut-off values of ratios (for T3) or the presence of T1 (for T4). In individuals with hypothyroidism where the tendency is towards the maintenance of homeostasis (as in the case of athletes), it was not possible to clearly detect the consumption.

Regarding to Endogenous Steroid Profile in urine, differences in the markers of the ABP steroidal module were found considering samples of athletes declaring and not declaring the supplementation with TH in the Doping Control Form. Although not all assessed parameters showed significant differences, there was observed a tendency to decrease steroid concentrations in the group of athletes who declared the consumption of levothyroxine. Female groups showed higher differences between declaring and not-declaring athletes' respect to males. The results for pregnanediol were a remarkable point, especially in the case of males. The information on TH therapy by athletes may be helpful for the correct interpretation of the ABP as happens for other markers considered confounding factors of the urinary steroid profile in the current WADA documents.

Code: ISF18E01JC 

The blood doping is a significant challenge in our fight to ensure fair athletic competition. The blood doping typically involved the transfusion of red blood cells which have been stored outside of human body. We have found that red blood cells have significant amount of RNAs which are not well characterized. Therefore, we will employ cutting edge single cell technology to identify whether subsets of RBCs and/or RNA will undergo storage-associated changes to distinguish these stored red cell from fresh red cells. We will develop single cell RNA-FISH methods identify individual stored red cells as a novel way to detect blood doping.

Main Findings: 

1. During blood storage, we have extensively validated the induction of miR-720 during storage. The miR-720 induction are consistent in all 15 tested samples and likely to comprise the stroage signature that can be used to detect ABT.

2. miR-720 is a cleave product from threonine-tRNA. During storage, there is concordant increase in miR-720 and remaining tRNA fragments based on Northern blots.

3. However, we noted that increased tRNA cleavage is not a general phenomenon. When we probed the small RNA Northern with differenet tRNA probes, we did not see the increased tRNA fragments seen for the miR-720. In addition, when we perform small RNA-Seq of fresh vs. stored RBC, we did not observe a general increase in tRNA fragments.

4. Small RNA-Seq of the fresh vs. stored RBC samples further reprodcued the induction of miR-720 by 16 folds. In addition, we identified additional transcripts which were increased by at least 10-fold during storage. We have reproduced the RNA-Seq in 4 additional paired samples and in the process of analyzing the data. Therefore, these storage-enriched transcripts can serve as robust storage signatures.

5. The stored RBC lysates contain the cleavage activities that include angiogenin, a stress-responsive nuclease. Angiogenin in the storage solution is increased during in vitro cleavage. The immune-depletion of angiogenin significantly reduced the miR0720 cleavage and addition of the recombinant angiogenin enhance the miR-720 induction during storage.

6. We have also optimized the single cell RNA-FISH methods to validate the increased miR-720 positive population during storages. This detection method can be used to detect small portion of RBC cells which have been stored and mixed with fresh RBCs.

7. It is also possible to separate fresh vs stored RBC cells based on opitcal volume changes stressed in microfluidic devices using quantitative phase imaging. Such methods can detect single stored red cells without labeling.

Code: 18C07KD 

Insulins are therapeutically used for the treatment of diabetes mellitus. There are rumors that insulins are used by athletes for anabolic properties. Consequently their use is prohibited by WADA. To control their abuse several sensitive methods for the detection of insulins in blood and urine have been described for doping control purposes. Generally, the applicability of these methods is illustrated with blood and urine samples from diabetic-patients. Indeed, blood and urine samples can be easily collected from diabetic patients without major ethical concern. Unfortunately, it can not be excluded that these samples are not representative for a healthy athlete population because of the diabetic status of the patients. Additionally, these spot-samples don’t give information on detection times. In general, no administration studies, from which the results are readily applicable to doping-control, have been performed. Therefore the aim of this project is to administer a single dose of insulins to healthy volunteers and to investigate detection times in blood and urine.

Because administration of a high dose of insulins can result in a life threatening situation only a low dose will be administered (0.05IU/kg). Three short acting insulins Lispro, Aspart and Glulisine will be investigated. Blood and urine samples will be collected from 1 week before administration, until 3 days after administration. The result of this project will be useful for doping organizations to set testing windows and for doping laboratories to evaluate their detection methods.

Main Findings: 

A simplified immunoaffinity purification LC-HRMS method was presented for the identification of the synthetic insulin analogue Lispro, Aspart and Glulisine in serum and urine samples. LODs obtained in serum for all 3 compounds was 500 pg/ml. In post administration serum samples, the insulins were detectable up to 6 hours.

More importantly, urinary detection resulted in much better results in terms of method development, validation (LODs 5 pg/ml, LOIs 10 pg/ml) and detection windows of these three rapid-acting insulins. Following the single injection (0.05 IU/kg), the administered analogues could be detected up to 12 hours and identified according to the TDICR2015 document up to about 9 hours using the presented analytical strategy.

Code: 18A28PA 

The aim of this project is to produce certified reference materials (CRMs) to ensure the accuracy and traceability of measurements of the stable carbon isotope ratios of steroids used to confirm Adverse Analytical Findings (AAF) in sports doping analysis. These CRMs will be used for validation of GC-C-IRMS methods and confirmation of Adverse Analytical Findings in accordance with WADA Technical Document TD2016IRMS. The ability of WADA-accredited laboratories to comply with this document is reliant on the availability of reference materials of appropriate steroids certified with traceable values for 13C isotope ratios. The availability of such materials is currently limited.

The proposed substances are boldenone, boldenone M1 and formestane.  Certified values for the δ13C values of each steroid and their associated measurement uncertainties will be determined by a combination of reference measurements with metrological traceability to VPDB made by NMIA using Elemental Analysis (EA-IRMS) and Gas Chromatography (GC-C-IRMS) Carbon Isotope Ratio Mass Spectrometry.

Main Findings: 

Two new CRMs have been prepared providing three steroids certified for stable carbon isotope delta values (δ13CVPDB). These materials have been designed to assist anti-doping laboratories to validate their calibration method for stable carbon isotope measurements of boldenone, boldenone metabolite and formestane to ensure accuracy and traceability in compliance with WADA Technical Document TD2019IRMS. The CRMs are packaged as dried steroids sealed in ampoules. MX020 consists of a mixture of boldenone and boldenone metabolite 1 and MX021 contains a single analyte, formestane.

Elemental Analyser Isotope Ratio Mass Spectrometry (EA-IRMS) was employed as the priamry reference method to assign δ-values for pure steroid starting materials due to the low uncertainty associated with this technique. Calibration was performed using a two-point normalization approach [2] which employed USGS-40 and IAEA-CH-7 as calibration standards, permitting traceability to the internationally recognized Vienna Pee Dee Belemnite (VPDB) carbon isotope reference standard. Potential bias in the assigned EA-IRMS values was investigated both through theoretical modelling using the steroid starting material’s purity data and through data obtained using the gas chromatography coupled to combustion isotope ratio mass spectrometry (GC-C-IRMS). Calibration and normalization of the GC-IRMS results were performed using the MX018 certified steroid standards with traceability to the VPDB reference. Homogeneity assessments of MX020 and MX021 were carried out using fifteen randomly selected ampoules. Stability of the CRMs were verified by analysis of randomly selected ampoules after storage at 4 °C for a period of upt to 5 months and at 40 °C for a period up to 30 days.

Code: ISF18R01AB 

In this project, we propose to perform additional validation of the EPO gene doping test with blood samples on the conditions for samples storage.  This includes factors affecting centrifugation and thawing, assay performance, improve separation of Tm peaks, storage temperature.  The assay will be validated with the optimal conditions.

The further validation of the EPO gene doping test will facilitate completion of the Technical Document for gene doping detection in blood and implementation of the test in the near future.

Main Findings: 

We performed additional validation of the polymerase chain reaction (PCR)-based erythropoietin (EPO) gene doping test using blood samples. We tested various conditions for prolonged sample storage and determined storage temperatures and duration, as well as sample processing prior to freezing, which do not compromise reliability of detecting the EPO doping gene in blood. We also improved test reliability by using a more suitable form of Uracil-DNA Glycosylase in PCR mix and tested other modifications to the test protocol with the intention to make the test easier and more legally defensible.
The results and outcomes from this project were incorporated in the Test Protocol. We conclude that the PCR-based EPO gene doping test using whole blood samples was fit to be implemented in doping control.

Code: 18A17MP 

The identification of anabolic androgenic steroids (AAS) is a vital issue in doping control. Due to the performance enhancing properties of AAS, the World Anti-Doping Association (WADA) banned their use but according to the annual report of WADA, steroids are still very popular amongst athletes and are responsible for 40% of all adverse analytical findings. The search for metabolites with longer detection times remains an important task and the introduction of new long-term metabolites for exogenous AAS such as for example stanozolol, methanedione and dehydrochloromethyltestosterone, led
to a 4 - 80-fold increase of adverse analytical findings due to the prolonged detection time.

Recently, our laboratory developed a new strategy for finding new long term metabolites. The approach uses low energy EI GC-QTOF and it is applicable to a wide range of different anabolic steroids. The technique has been tested in a proof-of-concept setting and the combination with a robust polar column allows the analysis of both derivatized and non-derivatized steroids, further expanding the application window and increasing the chances of finding new long term metabolites.

Main Findings: 

A search for new metabolites was performed by applying the low energy EI GC-QTOF product ion scan strategy. To increase the chances of finding new long-term metabolites this strategy was applied using different (complementary) approaches: Anaylses of both the derivatized and non-derivatized steroid form in the glucuronidated sulfated and free steroid fraction.

The main conclusion is that the strategy suffers from a lack of sensitivity that cannot be sufficiently compensated by the increased selectivity. Unfortunately, this meant that the procedue proved to be inferior to the MRM CI GC-MS/MS approach for detection of new metabolites (WADA project 16A01MP). Consequently, ne new metabolites could be found within this study and the approach failed to find many of the metabolites that were detected with the MRM CI GC-MS/MS detection protocol.

However, during this study we did acquired more knowledge on the analysis of sulfated steroids on GC-MS and this provided us with some new insides on their GC-MS behavious, prompting us to pursue a more in depth study of directly injecting non-hydrolyzed sulfated steroid on GC-MS. We are convinced that this direct injection strategy has great potential to, in the future, lead to the discovery of new long term metabolites and/or will allow the inclusion of long term sulfated metabolites in a general GC-MS initial testing procedure.

Code: 18C05CR

Chapter S2 of WADA’s Prohibited List 2018 (“Peptide hormones, growth factors, related substances, and mimetics”) lists EPO-Fc under sub-chapter 1.1 (“Erythropoietin-Receptor Agonists”). The current version of WADA’s technical document on ESA-analytics (TD2014EPO) describes general criteria of positivity for ESAs (e.g. rEPO, NESP, CERA). Since EPO-Fc cannot be directly detected by IEF-PAGE, SDS- or SAR-PAGE has to be used. However, chapter 2.1.2.4 "EPO-Fc" of the technical document does not define detailed criteria. The reason is that no data from human administration studies of EPO-Fc exist. Hence, it is unclear if both bands, which are typically observed for the standard (the strong band of the monomer AND the weak band of the dimer), have to be present for an adverse analytical finding.

So far, no approved EPO-Fc pharmaceuticals are available. Hence, administration of EPO-Fc to human test persons will be ethically not justifiable. For that reason we plan a study with rats. The test animals will receive EPO-Fc at a dosage, which can be still clearly detected after 48 hours in serum (160 μg/kg) according to literature.  Subsequently, serum and urine will be collected and tested for EPO-Fc by SAR- or SDS-PAGE. The study will help to clarify if (1) both bands of EPO-Fc are still observable after 48 hours of circulation in blood, and (2) EPO-Fc can also be detected in urine. Based on these results, more precise criteria for EPO-Fc might be specified in TD2014EPO.

Main Findings:

An administration study of EPO-Fc was performed with rats. After a sungle subcutaneous injection of 160 μg/kg EPO-Fc, serum and urine samples were collected and analyzed by SAR-PAGE after immunoaffinity purification. EPO-Fc could be clearly detected in just 10 μl of serum. No degradation products or interferences due to non-specific binding were observed. Both, the bands of the monomeric and dimeric EPO-Fc were detectable in all samples. EPO-Fc was also found in the urine samples, but at a muc lower concentration. However, additional bands were also observed, which might be degradation products of EPO-Fc. In conclusion, blood should be the preferred matrix for detecting EPO-Fc in doping control samples. The simultaneous detection of both bands (monomer and dimer) on SAR- or SDS-PAGE should pose the main criteria for the presence of EPO-Fc, when these methods are applied.

Code: 18B09CR

Chapter S4 of WADA’s Prohibited List 2018 (“Hormone and metabolic modulators”) lists myostatin inhibitors under sub-chapter 4 (“Agents modifying myostatin function(s)”). Follistatin (FST) suppresses signaling of myostatin and subsequently leads to an increase in muscle mass and loss of body fat. FST is a secreted glycoprotein, which can be found in many tissues and organs (e.g. pituitary, bone marrow, ovary, kidney, liver, blood vessels). Due to alternative splicing, three FST-isoforms exist (FS-288, FS-300, and FS-315). The isoform with 315 amino acids (FS-315) is the dominant one. FS-315 can also be detected in blood. Typical concentrations in serum and plasma are in the range of 2-3 ng/mL.  

So far, no approved follistatin pharmaceuticals are available.  Nevertheless, follistatins can be bought from many internet providers for “research purposes”. Their products are labelled either “Follistatin”, “Follistatin 344”, or “Follistatin 315”. Most of these proteins are expressed in E. coli and hence lack the characteristic glycosylation of human endogenous follistatins. This fact will be exploited in order to detect doping with follistatins. After immunoaffinity purification (serum/plasma, urine), FST will be separated by electrophoresis (SDS-, SAR-, or IEF-PAGE) and detected by Western blotting. Due to the missing glycosylation, “black market” FSTs will not only differ in molecular mass but also isoelectric point (pI) from the endogenous FSTs.

Main Findings:

Follistatin (FS), a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA Prohibited List 2022". While currently no approved pharmaceutical formulations of Follistatin are available, Follistatin can be bought on the black market. Most of the products are labelled "Follistatin 344" (FS344), few "Follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only 9 of the 17 tested products actually contained Follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Suprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomoers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western bloting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-FS antibody can be used. For immunoprecipitation (IP), a polyclonal anti-Follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practicall all currently available Follistatin standards were investigated. The detection limit of the method for back market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 μL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous Follistatin is possible.

Code: 18C02MT 

Testosterone misuse still remains a challenging task for doping control laboratories as this steroid is produced naturally by each and everybody. As it is detectable in all urine samples, concentration-based thresholds have been established to uncover testosterone administration. As soon as these thresholds are exceeded, samples are forwarded to isotope ratio mass spectrometry determinations (IRMS) to elucidate the steroid´s source and to unambiguously differentiate between naturally elevated testosterone concentrations and doping.

Recently a promising new target analyte for IRMS determinations was reported, epiandrosterone (EPIA). This steroid enables to prolong the detection of a single testosterone or testosterone prohormone administration from 24 h to more than 100 h using IRMS. Unfortunately, EPIA is only excreted into urine in it sulfoconjugated form while all other steroids routinely employed in IRMS are excreted glucuronidated. To investigate EPIA an additional time consuming step in sample preparation is inevitable.

The aim of this study is to investigate the potential of two other possible long term markers in IRMS excreted glucuronidated and therefore avoiding the additional step. The so called “Epidiols” are known long term metabolites of epitestosterone and might also work for other steroid administrations. In a preliminary study employing dehydroepiandrosterone both Epidiols were remarkably influenced by the steroid administration supporting the hypothesis that these new markers will improve steroid detection by IRMS. To further substantiate this finding we are going to re-analyze other excretion studies after improving and validating the already existing method for IRMS determinations of both Epidiols.

Main Findings: 

A novel IRMS method for determination of carbon isotope ratios (CIR) of 5a- and 5bEpiD was developed and fully validated in line with WADA requirements encompassing limits of detection, linear range and absence of isotopic fractionation. By means of investigations on a reference population encompassing n=72 individuals it was possible to derive referece-based decision limits, which demonstrated that the novel method is fit-for-purpose. Several administration trials were investigated to elucidate the potential of both markers. For a single oral T administration, the effect on both 5a- and 5bEpiB was less pronounced compared to traditional markers and offreed merely short detection windows. After consecutive transdermal T-Gel administrations, especially 5bEpiD was significantly influenced while 5aEpiD shows similar CIR,s as 5a- and 5bDIOL. Unfortunately, the first sample after cessation of T-Gel administration was collected after 5 days, and here all CIR values had returnedd to natural abundance. The administration of a single oral dose of androstenedione resulted in a prolonged depletion of CIRs for 5a- and 5bEpiD compared to other target compunds, but the influence was less pronounced, i.e. the CIR values were not found to be influenced beyond the established reference-based decision limits.

While a direct application of 5a- and 5bEpiD as long-term markers for steroid administrations appears to offer limited added value, these additional target compounds may be beneficial in detecting multiple transdermal T-Gel administration even if so called low-doses are applied. Moreover, they could support the differentiation between single administrations (including inadvertent exposure) and multiple and therefore intended administrations. Further research will be necessary to elucidate this potential of 5a- and 5bEpiD.