Methods to increase oxygen transfer International International Symposium Symposium Jordi Segura Accredited Antidoping Laboratory, Municipal Institute for Medical Research IMIM-UPF, Biomedical Research Park PRBB, Barcelona; IOC Medical Commission, Games Group, Lausanne, IOC Rome Rome , 14 , 14 october october 2006 2006 IMIM-UPF PRBB
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Methods to increase oxygentransfer
InternationalInternational SymposiumSymposium
Jordi SeguraAccredited Antidoping Laboratory, Municipal Institute for Medical Research IMIM-UPF, Biomedical Research Park PRBB, Barcelona;
IOC Medical Commission, Games Group, Lausanne, IOC
RomeRome, 14 , 14 octoberoctober 20062006
IMIM-UPF PRBB
Haemoglobin content and exercise
Ref. Ekblom B, Goldbarg AN, Gullbring B. Response to exercise after blood loss and reinfusion. J Appl Physiol 1972 Aug;33(2):175-80
-25
-20
-15
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0
5
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-30 -20 -10 0 10 20 30Δ Hb conc. %
Δ max VO2 %
• 1984 OG Los Angeles: first acknowledgement that blood transfusioncould be common practice in some sport disciplines (cyclism)
• 1994 OG Lillehammer: first control during OG of blood transfusion
• 1985 Amgen: Introduction of recombinant erythropoietin,
• 1998 Doping crisis: availability of EPO during the Tour de France
• 2000 First EPO detection methods: direct and indirect markers
OO22 solubilitysolubility atat 3737°°C (ml C (ml gasgas/100 ml /100 ml liquidliquid)) 33 5353COCO22 solubilitysolubility atat 3737°°C (ml C (ml gasgas/100 ml /100 ml liquidliquid)) 5757 210210
• PROPERTIES OF PFCs: PerflubronPerflubron®®H2O
LIPOSOME LIPOSOME EMULSIONEMULSION
LipidLipid bilayerbilayerPFCPFC
Ways to increase oxygen transport and delivery
• Blood transfusions• Modified haemoglobins• Allosteric modifiers of haemoglobin• Perfluorocarbons• rHuEPO, mimetics and analogues• Altitude training - hypoxic houses• Gene therapy with EPO genes
• BIOLOGICAL ACTIVITY
hEPO (natural)
BFU-E Burst-forming unit erythroid
GM-CSFIL-3
EPO
Stem cell(bone marrow)
CFU-E
EPO receptortransferrin receptor ( Tfr )
Colony-forming unit erythroid
proerythroblast
Fe (transferrin)Hemoglobinsynthesis
blood
Bone marrownormoblast reticulocyte
reticulocyte erythrocyte
sTfr
sTfr
enucleation
rhEPO
• PHARMACEUTICAL PRODUCT (recombinant):
- Recombinant product obtained by expressing the human EPO gen in different cell lines: CHO, BHK, etc.
- Commercially available since 1985 as “EPOETIN ALFA”
-many isoforms with different charges: MICROHETEROGENEITY
EPO
MarkerLine
• rh EPO DIRECT MARKERS (urine)
5.21
3.77
4.42pH
Standardof uhEPO
(sigma)
Std rhEPOβ uEPO in a real
Negative urinePositive urines
Std rhEPOα
Std rhEPOα+β Std
rhEPOω
Std uhEPO(NIBSC)
Lasne, de Ceaurriz, et al, 2000 Pascual , Segura, et al, 2004
Mimetics and analogues according to theWADA Prohibited List
• ANALOGUE
– An analogues is defined as a substancederived from the modification or alterationof the chemical structure of anothersubstance while retaining a similar pharmacological effect
NESP (Darbepoetin alfa): Hyperglycosilated rhEPO
• DIRECT MARKERS (urine) — SENSITIVE DETECTION —
NESPrhEPOα+β
rhEPOω
Std hEPO
(NIBSC)
pH 6
pH 2
Mimetics and analogues according to theWADA 2004 Prohibited List
• MIMETIC
– A mimetic is defined as a substances withpharmacological effect similar to that of anothersubstance, regardless of the fact that it has a different chemical structure
SEP (“Synthetic Erythropoiesis protein”)
• DEVELOPMENT: Gryphon Therapeutics (San Francisco, USA) Blood ReseachInstitute (Milwaukee, USA)
• CHEMICAL PROPERTIES:
- Chemically synthesized. - Nearly identical to the EPO
protein back-bone.- Substitute oligosaccarides by
a precision-length,negativelycharged polymers
Ways to increase oxygen transport and delivery
• Blood transfusions• Modified haemoglobins• Allosteric modifiers of haemoglobin• Perfluorocarbons• rHuEPO, mimetics and analogues• Altitude training - hypoxic houses• Gene therapy with EPO genes
2 groups:
NORM (N=8) training at sea level
HYPO (N=8) training at sea level with resting in the hypobaric chamber
16 highly trained male triathletes
Hypobaric chamber
participated in arandomised controlled trial
Are false positives possible by hypobaric hypoxia?
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1 8 15 22 29 36 43 50
Time (days)
Sim
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ted
alt
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(m)
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Time (days)
Sim
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(m)
HYPO group followed hypobaric exposure in resting conditions
NORM group remained in normobaric conditions
t8’ t33’
Blood and urinesamples collection
t8 t33 t46
Post 1 wk
Treadmill test
Pre
Field test
Post 2 wkPost
Outcome
Both ON/OFF scores and
endogenous EPO isoformswere slightly modified,
BUT NEVER ENOUGHto produce
any false positive result
3 Are false positives possible (eg. by hypobaric hypoxia?)
Segura J, Rodriguez F et al, 2005
Ways to increase oxygen transport and delivery
• Blood transfusions• Modified haemoglobins• Allosteric modifiers of haemoglobin• Perfluorocarbons• rHuEPO, mimetics and analogues• Altitude training - hypoxic houses• Gene therapy with EPO genes
Monkeys injected with a virus carrying the gene for EPO
Gene therapy with EPO
Conclusions
• Artifical increase of oxygen transport and release is a powerful formof doping in sport.
• Modified haemoglobins, perfluorocarbons and efrapoxiral will be easily detectable and probably will not represent a major real challenge.
• Doping substances have expanded towards engineered analoguesand mimetics of endogenous substances.
• New expertise has been incorporated to doping control to copewith the new challenges.
• Sophisticated methodology is needed to evidence thepresence of those substances in biological fluids, particularlyto differentiate synthetic or recombinant analogues from thenaturally occurring hormones.