London 2012: Holding back the Hematide

As preparations continue for London’s moment in the sporting spotlight next year - Dermot Martin takes a look at drug testing in sport and highlights a new performance enhancer that could hit the market before the 2012 Olympics

Catching athletes who rely on performance enhancing drugs is like taking part in a cycle pursuit race: two cyclists start out from the opposite sides of an oval track and attempt to catch their opponent within a specified distance. In the murky world of doping, the cheating athlete is usually some distance ahead of the researchers who are frantically playing catch up.

Chemists at the World Anti-Doping Agency (WADA) have faced many hurdles developing a universally accepted testing regime for a panoply of performance enhancing substances such as nandralone and stanazolol made infamous by Canadian sprinter Ben Johnson.

In recent years they have battled the emergence of genetically engineered human hormones, chiefly recombinant human erythropoietin, (rhuEPO). There has been much criticism in some circles at the quality of the test for EPO and rhuEPO and some athletes caught out by testing have even tried but failed in legal challenges.

Where does  EPO testing stand as London 2012 approaches? The hormone became the fashionable choice of performance enhancing substances among athletes determined to cheat their way to fame and fortune in the mid-90s. It boosts the body’s ability to create red blood cells and hence the blood’s oxygen carrying capacity. Before the 2004 Olympics there was no reliable testing regime so the development of a test in time for Athens is still regarded by many as WADA scientists’ finest hour.

That breakthrough was made by Lasne and de Ceaurriz in 2000 at the French national anti-doping laboratory. They used immunoelectorphoresis (monoclonal anti-body reaction to rhuEPO) and double blotting (IEF/DB). It heralded the first recognised test and in 2002 Don Catlin – the anti-doping guru who is the nemesis of all cheating athletes – announced the first successful detection of EPO abuse. It was thought to be the death knell for rhuEPO as a performance enhancer although it continues to be invaluable in the medical field.

Testing relies upon isoelectric focussing (IEF) methods, also known as electro-focusing. Familiar to anyone who uses electrophoresis, it is the technique common in analytical laboratories and is a type of zone electrophoresis – usually performed in a gel – which takes advantage of the fact that a molecule's charge changes with the pH of its surroundings. Endogenous EPO and recombinant heap migrate differently in an electric field.

Rumours began to circulate in 2005 that all was not well with the test. Thirty four labs around the world are accredited with testing for EPO by WADA, but in order to confirm the viability of the test, two centres were chosen for a special study to confirm its effectiveness1. The labs embraced the study, or so it seemed, but once the results were reported it caused a small earthquake in the testing community, the aftershocks of which are still being felt. The study threatened to derail confidence in WADA’s ability to trap the EPO cheats.

The framework of the study was simple: there were two laboratories labelled A and B and eight male volunteers, who for the purposes of the study were not recognised athletes. The volunteers were injected with 5,000 IU rhEPO every second day for 14 days in what was known in the trial as the “boosting period”. For the following two weeks, called the “maintenance period”, they received one injection every seven days. The idea was to try to create the conditions under which an athlete might employ the hormone.

Before the injection, bloods were drawn and on eight other separate occasions. Urine sample were taken before each blood sampling and in addition on six other days. The volunteers were invited to pedal a bicycle ergometer prior to the injections and on three other occasions. The value of recombinant EPO to an athlete is that it increases the oxygen carrying capacity of his/her blood and the results showed that the injections certainly enhanced the performance.

But the problems started when the results from Lab A and B were compared. Lab A tested the samples collected during the “boosting phase” and concluded that they were all positive for ruEPO. Identical samples tested by Lab B suggested – according to the study co-ordinators – that no hormone was present.

Furthermore samples collected during the maintenance period analysed by Lab A determined that six out of 16 samples were positive and two were “suspicious”.  Lab B found no positive results.

In the post treatment phase, Lab A found only two samples were positive and Lab B found all 24 samples were negative. Alarm bells started ringing. If these result had any meaning at all it was that athletes only faced a small risk of being tested positive. Indeed if the samples were analysed by Lab B it would appear there was no risk of getting caught at all.

The identity of Labs A and B was never revealed but there was a flurry of indignation as soon as the study’s findings were made public from the WADA accredited laboratory in Cologne, Germany. It was led by its director Dr Wilhelm Schanzer, who was merciless in his criticism of the study and remains firmly of the belief that the IEF testing method is working for EPO. Dr Schanzer effectively demolished the basis of the study saying there had been a total breakdown of communication from start to finish.

Polymer chemists will be familiar with the technique of PEGylation. It is an established drug delivery system whereby the synthetic polymer is bonded chemically to a protein peptide or nucleotide. The combination is often called a conjugate. PEG itself is a linear polymer with a regular repeat unit of the general structure: (CH2CH2O)n-. The most useful kind of PEG has a methoxy group on one end and a hydroxy group on the other. The average molecular weight is between 5kDa and 40kDA – that is about 100 to 1,000 –(CH2CH2) – repeat units all in a line. However the nature of synthetic polymers is the polymerisation of monomer to polymer almost always results in a distribution of actual chain lengths. This distribution is known as polydispersity or molecular weight distribution. Polymerisation of the monomer to polymer almost always results in other imperfections such as diols. There are PEG chains that have hydroxyl groups on both ends and are typically almost double the average molecular weight in size.

Schanzer said: “Before Lab B agreed to participate in the study, it requested assurance that it had been approved by an anti-doping authority and that the samples were not originating from athletes. Lab B was never informed about any intended inter-laboratory comparison and subsequent publication of the data.

“The lab issued a statement that the samples were measured by a novel new approach at that time, the SDS-Page analysis (sodium dodecyl sulphate polyacrylamide gel electrophoresis).”

In other words Lab B didn’t apply the same testing method as Lab A because it wasn’t aware that its results were to be used in any way to focus on the quality of the testing. It was more interested in testing the newer method.

It did not apply the entire IEF method used at Lab A. This entire method involved screening analysis, repetition analysis including different tests for activities that can yield false positive results, and inspection of the results by a second laboratory.  As Dr Schanzer said the quality of Lab B results had to be judged on that basis: “The use of abbreviated screening is a major reason why no negative or positive results were reported but only unconfirmed as suspicious test results.”

He complained that the study authors should have requested a statement of the results at the proof reading stage of the manuscript and these misunderstandings would have been eliminated immediately. The main result of the publication is out right false said Schanzer.

“I am confident with the IEF method as critical points of the method such as active urine samples and changes of EPO bands following high exercise intensity (effort urines) can be controlled and ruled out by the new SDS-Page method. Additionally the SDS-Page method can be used for supporting evidence of positive samples and can be used to solve possible positive ‘borderline’ cases in IEF analysis, he said”

His explanation rings true even though there have been other doubts raised about false positives using IEF2. At the Department of Clinical Chemistry, University Hospital, Ghent, Belgium, Monique Beullens, and Joris R Delanghe experienced what they described as a false positive test in urine sample from a subject who displayed a regime which was ‘post-exercise’, protein-rich urine.

They theorised that this was probably because the adopted monoclonal anti-EPO antibodies used setting up the test were not monospecific for rhuEPO. But Don Catlin himself took issue with that piece of research.

In a letter to the journal Blood he wrote: “We take issue with the use of the term ‘false positive’. If the compound detected can be identified as not rhuEpo, then it cannot cause a false positive.

“This term sensationalises an otherwise interesting case report that could in due course contribute to the body of science.  The criteria used by the WADA laboratories are well known and readily available. Beullens et al do not state the criteria they used to make the "false-positive" claim.”

Catlin wrote that using the WADA  criteria, the "false-positive" electropherogram they published is clearly negative. He points out that the pH range of the test is not clear and without that the data cannot be fully interpreted. To be fair to the Belgium researchers they had no real doubts about the efficacy of the urine test for EPO derivatives.

The force remains with the IEF test for EPO and rhuEPO. By 2006 about 6,800 urine samples had been through the procedures including 2,600 for doping control of atheletes. Nine positive cases for rhuEPO had been found and even the use of advanced version with an extended half life (CERA) led to the expulsion of Tour de France cyclists.

That EPO testing is satisfactory is shown by proof the decline in detections. The athletes themselves are saying: “Yes –good test, too good for us to risk taking rhuEPO”.  That is perhaps the best unspoken compliment that could be paid to the scientific detectives out to trap the cheats.

One newer derivative called Hematide made by Affymax recently came through a stage 2 trial in the US as a drug to aid anaemia. It could be on sale in the US by 2012 but it’s likely it is already available on the black market. To date there is no testing regime for this substance even though it operates biologically and physiologically like EPO boosting the blood’s oxygen carrying capacity.

Anti-doping authorities are always strategically vague when talking about whether a test is already in place for developing drugs, not wanting cheats to know what solutions have been found or perfected. The downside of the strategy is that it leads to speculation about the quality of the tests as we’ve seen here with EPO.

Hematide is a synthetic peptide, substituted with PEG or polyethylene glycol (thus described as "PEGylated").  It is stable at room temperature and lacks cross-reactivity with anti erythropoietin antibodies which are key to the testing regime. It has long duration action and allows for once a month dosing, but for the analyst it differs from EPO and ruEPO because it is a PEGylated peptide.

The end of the year is the target date for a verifiable test. One leading scientific advisor to the French Anti-Doping Agency (AFLD), Michel Rieu said: “Hematide is a form of EPO which I believe is being widely used. Progress [in testing] had been delayed because WADA first had to come to an agreement with the laboratory [developing hematide] so that the molecule can be provided to the [anti-doping] laboratories so that a testing method could be implemented."

The two laboratories involved are in Lausanne (Switzerland) and Chatenay-Malabry (France). Rieu is adamant: “There is a very good chance that Hematide will be detectable this year,” he said. The sports administrators will be relieved, but watching with interest.

Hematide is already specifically named on the banned list, said Gary Wadler who leads the WADA committee that determines the banned-substances list.

Earlier this year, WADA signed an agreement with a group representing pharmaceutical companies that gets the companies to voluntarily share information with drug police when they're developing new products. It was considered a breakthrough, giving anti-doping authorities a window into what's out there. In return, WADA has to agree not to share proprietary information between competing companies.

There are many forces at work in this war of attrition against the cheats. The analysts are definitely sensitive about revealing much in public about their testing regimes. They prefer to keep the athletes on the wrong foot. The companies which are testing their products in the medical sense a fearful that they may lose commercial advantage to competitors if details of structures and clinical trial outcomes become widely exposed.

In any war intelligence and secrecy are two ingredients that keep antagonists on their toes. Hopefully the cheats will be deterred by the determination and ingenuity of the scientists but best not hold your breath!

The Author: Dermot Martin

References: 1. J Appl Physiol 2008 10 1152 2. Blood, 15 June 2006, Vol. 107, No. 12, pp. 4711-4713.