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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
-10
-5
0
5
10
15
20
25
30
-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
• 1990’s UCI and FIS: Indirect markers (health prevention)• hematocrit• hemoglobin
• 2001 Masking agents: Plasma expanders HES (hydroxy-ethyl starch)
• 2000 RSR13 : Giro of Italy, one participant had available RSR13,first evidence that haemoglobin modifiers maybemisused in sport practice
• 2002 Darbepoetin (NESP) used at OG Salt Lake City
• 2004/2006 Transfussions OG Athens and Vuelta. Puerto affair.
World Anti-Doping Agency (WADA)
Level 1:
TestingLevel 2:TUE List
International Standards
Level 3:
Models of Best Practice
Rules andRegulations: IFs, NADOs, NOCs, EOs
ResultsManagement
EducationPrograms
Labo-ratory
NationalAnti-
DopingPrograms
World Anti-Doping Code
The World Anti-Doping CodeThe 2005 Prohibited List
PROHIBITEDSUBSTANCES• S1. Anabolic Agents • S2. Hormones and Related
Substances• S3. Beta-2 Agonists • S4. Agents with anti-
oestrogenic activity • S5. Diuretics and other
masking agents• S6. Stimulants• S7. Narcotics• S8. Cannabinoids• S9. Glucocorticosteroids
PROHIBITED METHODS• M1. Enhancement of
oxygen transfer• M2. Chemical and physical
manipulation• M3. Gene Doping
SUBSTANCESPROHIBITED INPARTICULAR SPORTS• P1. Alcohol• P2. Beta blockers
The World Anti-Doping CodeThe 2005 Prohibited List
• S2 Hormones and related substances– Erhytropoietin
• S5 Diuretics and Masking Agents– Plasma expanders
• M1 Enhancement of Oxygen transfer– Blood doping (autologous, homologous or heterologous
blood or red blood cell products)– Enhancers of uptake, transport or delivery of oxygen
(perfluorochemicals, efaproxiral, modified haemoglobinproducts)
• M3 Gene Doping
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
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
Hamilton blood tests show 'inconsistencies'By Andrew Hood
September 20, 2004
Tyler Hamilton is denying media reports of blood tampering that have been
detected in two samples taken since he won the Olympic time trial gold medal
last month.
The Vuelta a España was rocked overnight following reports that the UCI
informed Phonak team doctor Iñaki Arratibel that blood samples taken Aug. 19
and Sept. 18 showed traces of mixed blood cells. Follow-up tests were
scheduled for later Tuesday.
Phonak confirmed those reports, but said Hamilton has denied any
wrong-doing. Phonak officials have scheduled a press conference at the
team's headquarters in Zurich on Tuesday afternoon.
Old methods revisited
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
HBOCs (Haemoglobin Based Oxygen Carriers)
CrosslinkedCrosslinked polyHbpolyHb
CrosslinkedCrosslinkedtetramerictetrameric HbHb
Recombinant Recombinant HbHbαα1 1 andand αα2 2 fusedfused
ConjugatedConjugated polyHbpolyHb
TetramericTetrameric HbHb
ConjugatedConjugated HbHb
α1α2
β2β1α1 α2
β2β1
α1 α2
β2β1
α1 α2
β2β1
α1 α2
β2β1
α1 α2
β2β1
α
β β
Fe||
Fe||
Fe|| Fe||
α
α1 α2
β2β1
α1 α2
β2β1
α1 α2
β2β1EncapsulatedEncapsulated HbHb
α1 α2
β2β12,3-DPG
α1 α2
β2β12,3-DPG
α1 α2
β2β12,3-DPG
α1 α2
β2β12,3-DPG
α1 α2
β2β12,3-DPG
α1 α2
β2β12,3-DPG
bis(3,5 dibromosalicyl)fumaratepyridoxal-5-phosphateo-adenosine-5'-triphosphate
glutaraldehydeo-adenosine
polyoxoethylenepolyethylene glycoldextran
OxygentOxygentTMTM
PolyHemePolyHemeTMTM
HBOC 201HBOC 201TMTM
HemolinkHemolinkTMTM
HemAssistHemAssistTMTM
PHPPHPTMTM
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
RSR-13 (EFAPROXIRAL)• Allosteric modifier of Hb
• Increases maximum oxygen uptake (VO2 max)
• High urinary concentrations
(Breidbach, Catlin et al. 2001; Ventura, Segura et al. 2003)
N
H
OO
O
OH
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
PFCs (Perfluorocarbons)
• Synthetic hydrocarbon analogues. High solubility of O2
e.g. “PERFLUBRON” (perfluoro-octyl bromide)CCFF33--CCFF22-- CCFF22-- CCFF22-- CCFF22-- CCFF22-- CCFF22-- CCFF22BrBr
BoilingBoiling point (point (°°C)C) 100100 143143
DensityDensity atat 2525°°C (g/ml)C (g/ml) 11 1,931,93
ViscosityViscosity (centistokes (centistokes atat 2525°°C)C) 11 1,101,10
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”
- Epoetin Alfa“glycoform profile ALFA”
EPOADE® (Sankyo)EPOGEN® (Amgen)EPREX® (Jansern-Cilag)ESPO® (Kirin)GLOBUREN® (Dompé)PROCRIT® (Ortho Biotech)EPOPEN® (Esteve)
- Epoetin Beta“glycoform profile BETA”
EPOCH® (Chugai)EPOGIN® (Chugai)MAROGEN® (Chugai)RECORMON® (Boehringer M.)ERANTIN® (Boehringer M.)NEORECORMON® (Roche)EPOPEN® (Pensa)
- Epoetin Omega“glycoform profile OMEGA”
EPOMAX® (Elanex)HEMAX (Elanex)
- Epoetin Delta“glycoform profile DELTA”
DYNEPO® (Aventis-TKT)
-76.96 +299.3 RetHct +78.67 Hct +0.946 EPO +0.892 sTfr +2.08 %macrocytes
- ON MODEL :ON score = -12.40 +
-1566 RetHct +50.90 Hct +
-0.765 EPO
- OFF MODEL :OFF score =
rhEPO INDIRECT MARKERS
(Parisotto et al. Haematologica 2000; 85: 564-572)
Hb + 9.74 ln (EPO)- ON MODEL :
ON score =- OFF MODEL :
OFF score = Hb – 60 (ret%)1/2
(Gore CJ et al. Haematologica 2003; 88: 333-344)REEVALUATION OF MARKERS:
- Glycoprotein (mw: ~ 30 kDa) polipeptIdic chain 60%Glicosidic chains 40%
· 4 glycosidic chains: Asn 24, 38, 83Ser 126
CHEMICAL PROPERTIES
-different sugar composition
-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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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
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.