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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

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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

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


• Blood transfusions• Modified haemoglobins• Allosteric modifiers of haemoglobin• Perfluorocarbons• rHuEPO, mimetics and analogues• Altitude training - hypoxic houses Gene therapy with EPO

genes

α α

β β

Fe||

Fe||

Fe|| Fe||

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

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

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

• 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)

(Parisotto et al. 2000)

rhEPO INDIRECT MARKERS

blood

serum

-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:

Electrophoreticdetection in urine

Wide et al., 1995

rhEPO DIRECT 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

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

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.

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