Hormona del Crecimiento y Dopaje Genéticocmedica.coe.es/.../$FILE/9_Segura.pdf3 The World...

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Hormona del Crecimiento y

Dopaje Genético

Jordi SeguraLaboratorio Acreditado AMA, IMIM-Hospital del Mar,

Parque de Investigación Biomédica PRBB, Barcelona;

IOC Medical Commission, Games Group, Lausanne, IOC

• La llegada de Técnicas recombinantes ha revolucionado el abanico de sustancias dopantes

• Ha permitido el uso de proteínas y hormonas para curar enfermedades pero también su abuso en el deporte: EPO, hGH, LH, etc

• Ha propiciado el desarrollo de la Terapia Genética, pero al mismo tiempo la amenaza de su uso fraudulento en el ámbito deportivo

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Detección de substancias prohibidas:

1. Substancias exógenas: - Relativamente fácil, ya que se detectan compuestos

que el propio cuerpo no produce:

2. Substancias de estructura igual a las endógenas:- Mayor complicación por la necesidad de distinguir

el origen endógeno del exógeno

Control antidopaje – Dificultades

Detección de substancias de estructura igual a las endógenas:

Marcadores directos:

- Diferencias químicas sutiles entre la droga administrada y la hormona natural producida por el organismo

- Difícil obtener claros marcadores directos

Marcadores indirectos:

- Efectos fisiológicos

- Estudios poblacionales: probabilidad

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The World Anti-Doping CodeThe 2008 Prohibited List

PROHIBITEDSUBSTANCES• S1. Anabolic Agents • S2. Hormones and Related

Substances• S3. Beta-2 Agonists • S4. Hormones

anatagonists and modulators

• 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

S2. HORMONES AND RELATED SUBSTANCES (I)

The following substances, including other substances with a similar chemical structure or similar biological effects, and their releasing factors, are prohibited:

1. Erytropoietin (EPO)2. Growth hormone (hGH), Insulin-like Growth Factors

(e.g. IGF-1), Mecano Growth Factors (MGFs)3. Gonadotrophins (e.g. LH, hCG), prohibited in males

only4. Insulin5. Corticotrophins

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Detección indirecta de la GH

Proyecto GH 2000/4

Metabolismo hepático:• IGF-I• IGFBP-2• IGFBP-3• ALS

Metabolismo óseo:• Osteocalcina• P-III-P• PICP• ICTP

De entre ellos se buscaron marcadores sensibles al tratamiento con rhGH, pero relativamente inalterables con el tiempo o en respuesta al ejercicio.

Detección indirecta de la GH

-3 -2 -1 0 1 2 3 4 5 6 7

EM1_scal

Fig 5. Dotplots of scaled derived marker EM1, by day

d a y

0

21

28

30

33

42

84

Modelos obtenidos para la detección del abuso de rhGH

EM1=-2.269+0.7270·ln[P-III-P]+0.521·ln[IGF-I]

5

0

100

200

300

400

500

10 20 30 40

Age (years)

[IG

F-I]

(ng/

ml)

N=177

0

5

10

15

20

10 20 30 40

Age (years)[P

-III

-P] (

ng/m

l)

N=173

Effect of Age on GH-Responsive Markers

edad737.1011EM031.4560.2b1EM −⋅+=

Deportistas del CAR S.Cugat

• Implementación de la detección indirecta de hGH pendiente

• Diferentes métodos ofrecen diferentes resultados, aunque existen coeficientes de comparación

• Se busca reactivos de aplicación universal• Dependencia de la edad• Serán útiles para desarrollar un control

“inteligente”

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Detección de substáncias de estructura igual a las endógenas:

Marcadores directos:

- Diferencias químicas sutiles entre la droga administrada y la hormona natural producida por el organismo

- Difícil obtener claros marcadores directos

Marcadores indirectos:

- Efectos fisiológicos

- Estudios poblacionales: probabilidad

Gene Expression of GH Isoforms

transcription46

hGH mRNAimmature

-26-24 -23 31 32 71 72 126 127 191

splicing

22K-GH mRNAmature

20K-GH mRNAmature

1 2 3 4 5

1 2 3 4 5alternative splicing

hGH-N gene5‘ 3‘

Exon I II III IV V

B C DIntron

AB’

- 50Kb

5‘hGH-N hPL-1 hPL-4 hGH-V hPL-3 enhancerhGH gene cluster

Chromosome 17 (17q22-24)

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22K-GH vs 20K-GH

GH

HRP

anti-20K-GH

anti-GH

GH

HRP

anti-22K-GH

anti-GH

- Secreción normal de hGH:

-Nivel de fondo bajovariable- Picos intermitentesde gran magnitud de ambas isoformas

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22kDA hGH and 20kDa hGH following injection of0.2 IU/kg rhGH

Leung et al, Am. J. Physiol Endo Metabs, 283:836-843

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Human growth hormoneHuman growth hormone

191 aa – 22 kDa ~ 90 %176 aa – 20 kDa ~ 10 %

43 aa – 5 kDa (sequence 1-43)1

148 aa – 17 kDa (sequence 44-191)2

191 aa ? – 24 kDa (glycosylated)3

191 aa ? – 34 kDa (glycosylated)4

? – 12 kDa (glycosylated)4

Nα-acetylated formDeamidated form: Asp137/Glu152

Monomers, Dimers, Oligomers, Complexes

hGHhGH--N gene N gene –– pituitary glandpituitary gland

Immune cells Immune cells

Pituitary gland Pituitary gland

hGHhGH--V geneV gene

SyncytiotrophoblastSyncytiotrophoblastPlacenta Placenta

191 aa 22 kDa1 J.Immunol.Meth. 1998, 215, 1792 Trends Endocrinol.Metab. 1992, 3, 1173 Biochem.Biophys.Res.Commun. 1996, 228, 5494 Horm.Res. 2000, 53, 40

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HUMAN GROWTH HORMONE –Wu, Bidlingmaier & Strasburger, Munich University Hospital

rhGH InjectionOne isoform only:22kDA

Natural hGH SecretionMultiple isoforms:22kDa, 20kDa, 17kDa and others

Basis of a rhGH Doping Test1. Natural hGH is a mixture of types (isoforms), named for their relative sizes2. rhGH has only one GH isoform (22kDa)3. After injection of rhGH, the 22kDa isoformdominates in blood, and the other forms are missing. 4. Develop two GH tests:

Test 1: Pituitary GH Test (PitGH)Measures all the forms of GH made by the pituitary gland. Detects monomeric 22kDa but weakly

Test 2: Recombinant GH Test (RecGH)Measures only 22kDa

Doping or Not?Calculate the ratio GH test 2 (RecGH)

GH test 1 (PitGH)An increased ratio indicates dopingwith recombinant human hGH

Método directo para hGH• Método empleado en los Juegos Olímpicos de

Beijing• Empieza a ser utilizado de manera general• Limitaciones:

– Necesario recoger sangre– Corta ventana temporal de detección– Importante controles por sorpresa fuera de

competición• No se han detectado resultados positivos hasta

ahora

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Pero estas sustancias pueden también llegar a ser producidas “in vivo”

mediante la inserción de genes apropiados

Alerta sobre Dopaje Genético

• 2001: Gene Therapy Working Group, convened by the IOC-MC meeting:”Genetherapy and its future impact on sports”:

• Inclusion List, January 2003

WADA position

“the non-therapeutic use of cells, genes, genetic elements, or the modulation of gene expresion, having the capacity to enhance athletic performance, is prohibited in sport”

Probably not yet an actual problem. Gene therapy still mainly in clinical trials, but…

…what to do towards London Olympics?

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Degrees of potential geneticinterevention

• Genes and sports– Roger Bannister, first men to run one mile under 4 min.:

“Athletes are not born equal”• West African: short distance• East African: marathon• Caucasians; swimming

• Genetic screening: childs evolve top athletes• Genetic manipulation: gain genetic predisposition

• Marion Jones, Tim Montgomeny: child, genetic advantage?• Steffi Graf, Andre Agassi: child, genetic advantage?

• Gene doping: Gain advantatge through gene transfer

The World Anti-Doping CodeThe 2008 Prohibited List

PROHIBITEDSUBSTANCES• S1. Anabolic Agents • S2. Hormones and Related

Substances• S3. Beta-2 Agonists • S4. Hormones

anatagonists and modulators

• 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

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• Gene Therapy– 3000 patients, some (few) side effects. Trials

on going.• severe combined immunodeficiency disease• adenosine deaminase deficiency • haemophilia B• …..

– Few registered products so far: Vitraene(antisense technology); tumor supress gene p53 (reduce tumor growth; China); …

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Sport authorities Concerns

• 2001: Gene Therapy Working Group, convened by the IOC-MC meeting:”Genetherapy and its future impact on sports”: Inclusion List, January 2003

• 2004 / 2005 WADA Conferences: • 1st Conference at Banbury• 2nd Conference in Stockholm

Stockholm declaration• Gene therapy now represents a proven, although very immature and still

experimental field of human medicine.

• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.

• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.

• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .

• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.

• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.

• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.

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Potential Sport Targets

• GH / IGF-1 Promote muscle mass• Myostatin (negative regulator of muscle

formation). Myostatin blockers increase skeletal muscle.

• EPO Gene therapy with EPO increases haematocrit in animals more than 80%

• VEGF (Vascular endolthelial growth factor): Increase blood supply

• Endorphins (for pain)

Laboratory mice transferred with IGF-1 gene at the University of Pennsylvania seem to holdthe “beneficial” effects of IGF-1 throughouttheir lives.

Growth Hormone / IGF-1

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A german boy had mutation with lack of myostatin production

(Schuelke M, N Eng J Med 2004, 350: 2682-2688)

Transgenic rats

In 1997, scientistsMcPherron and Lee revealed to thepublic the ‘secret’of an anomaly thatlivestock breedershave capitalizedsince the late 1800’s: the gene responsible for bigbeefy cows .

Myostatin inhibition

CMV mEpo SV40 Neo

Scheme of the mEpo vectorused in our animal gene transfer studies

Genes expressing rEPO

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Awaiting for gene transfer Anaesthesia Tibialis muscle exposed

Muscle width measurementPlasmid injection Applying a conductive gel

Setting electrical parameters Electroporation Work done!

Hematocrit

50

55

60

65

70

75

80

85

90

2 8 14

Time (days)

HC

T%

Control Transfected

0

2000

4000

6000

8000

10000

12000

14000

0 2 4 6 8 10 12 14 16

Time (days)

[EP

O] m

U/m

l

Serum EPO

Effects after gene transfer to mice muscle

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Monkeys injected with a virus carrying the gene for EPO

Gene therapy with EPO

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Possible strategies for detectiongene doping

Screening• Detect plasmid vector sequences and immune response to viral

vectors• Differentiate transgenic DNA from genomic DNA• Microarrays to detect changes in gene expression• Proteomics to detect changes in gene expression• Indirect physiological models• Different isoforms (promising for EPO)• Others

Possible strategies for detectiongene doping

Screening• Detect plasmid vector sequences and Iimune response to viral

vectors• Differentiate transgenic DNA from genomic DNA• Microarrays to detect changes in gene expression• Proteomics to detect changes in gene expression• DNA bar codes (difficult, it depends on multiple parties)• Indirect physiological models• Different isoforms (promising for EPO)• Others

Confirmation• Confirmation by non invasive imaging detection of

unexpected expression in an ectopic tissue

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IMAGENENon invasive molecular imaging of gene expression useful for doping control: Pilot study in animals after erythropoietin gene transfer

You may say I’m a dreamer, but I’m not the only oneImagine, John Lennon, 1974RESEARCH HYPOTHESIS AND OBJECTIVES

- The gene transfer processes may produce the expression of mRNA for the target hormone-protein in unusual cells or tissues.

Cap

AAA AAA

Cap

- mRNA molecules will hybridize with suitable antisense modified oligonucleotides available to the tissue expressing the ectopic hormone-protein.

- If a label of appropriate energy is associated to the modified oligonucleotides, detection of the unusual hybridization may be carried out non-invasively in real-time by suitable imaging technologies.

Labeled TAT-PNA preparation Quality control

Anaesthesia Preparing administration I Preparing administration II

Administration Image adquisition I Image adquisition II

Chromatograms

Imaging

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Final Aim : Sensitivity and Resolution of PositronEmission Tomography (PET) or Single Photon EmissionComputerized Tomography (SPECT)

PETfacility

Micro PET facility

CONCLUSIONES

- La detección óptima de la administración de hormona de crecimiento debería integrar marcadores directos e indirectos

- El Dopaje Genético en el deporte está prohibido por razones éticas, tanto médicas como deportivas, y de prevención de riesgos.

- Debe incrementasre el apoyo a la investigación para el desarrollo de métodos de detección del dopaje genético en el deporte, a ser posible de aplicación relativamente simple.

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