Hormona del Crecimiento y Dopaje Genético

Hormona del
Crecimiento y
Dopaje Genético
Jordi Segura
Laboratorio 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
1
Control antidopaje – Dificultades
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
Detección de substancias de
estructura igual a las endógenas:
Marcadores indirectos:
- Efectos fisiológicos
- Estudios poblacionales: probabilidad
Marcadores directos:
- Diferencias químicas sutiles entre la droga administrada
y la hormona natural producida por el organismo
- Difícil obtener claros marcadores directos
2
The World Anti-Doping Code
The 2008 Prohibited List
PROHIBITED
SUBSTANCES
• 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
SUBSTANCES
PROHIBITED IN
PARTICULAR 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
only
4. Insulin
5. Corticotrophins
3
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
Modelos obtenidos para la detección del abuso de rhGH
EM1=-2.269+0.7270·ln[P-III-P]+0.521·ln[IGF-I]
Fig 5. Dotplots of scaled derived marker EM1, by day
day
84
42
33
30
28
21
0
-3
-2
-1
0
1
2
3
4
5
6
7
EM1_scal
4
Effect of Age on GH-Responsive Markers
Deportistas del CAR S.Cugat
500
20
N=177
[P-III-P] (ng/ml)
[IGF-I] (ng/ml)
400
300
200
100
0
N=173
15
10
5
0
10
20
30
40
10
Age (years)
EM1b = 2.560 + 4.031 ⋅ EM1 −
20
30
40
Age (years)
101.737
edad
• 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”
5
Detección de substáncias de
estructura igual a las endógenas:
Marcadores indirectos:
- Efectos fisiológicos
- Estudios poblacionales: probabilidad
Marcadores directos:
- Diferencias químicas sutiles entre la droga administrada
y la hormona natural producida por el organismo
- Difícil obtener claros marcadores directos
Gene Expression of GH Isoforms
- 50Kb
hGH gene cluster
5‘
hGH-N
enhancer
hPL-1
hPL-4
hGH-V
hPL-3
Chromosome 17
(17q22-24)
B’
Intron
hGH-N gene
A
5‘
Exon
I
-26-24
hGH mRNA
immature
B
C
II
III
-23 31
46
32 71
3‘
D
IV
V
transcription
72 126
127
191
splicing
22K-GH mRNA
mature
1 2
3
4
5
alternative splicing
20K-GH mRNA
mature
1 2 3
4
5
6
22K-GH
vs
20K-GH
- Secreción normal de
hGH:
-Nivel de fondo bajo
variable
- Picos intermitentes
de gran magnitud de
ambas isoformas
HRP
HRP
anti-GH
anti-GH
GH
anti-20K-GH
GH
anti-22K-GH
7
22kDA hGH and 20kDa hGH following injection of
0.2 IU/kg rhGH
Leung et al, Am. J. Physiol Endo
Metabs, 283:836-843
Human growth hormone
hGHhGH-N gene – pituitary gland
Pituitary gland
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
Immune cells
Nα-acetylated form
Deamidated form: Asp137/Glu152
Monomers, Dimers, Oligomers,
Complexes
Syncytiotrophoblast
Placenta
1
J.Immunol.Meth. 1998, 215, 179
Trends Endocrinol.Metab. 1992, 3, 117
3 Biochem.Biophys.Res.Commun. 1996, 228, 549
4 Horm.Res. 2000, 53, 40
2
hGHhGH-V gene
191 aa 22 kDa
16
8
HUMAN GROWTH HORMONE –
Wu, Bidlingmaier & Strasburger, Munich University Hospital
Basis of a rhGH Doping Test
Natural hGH Secretion
Multiple isoforms:
22kDa, 20kDa, 17kDa
and others
1. Natural hGH is a mixture of types
(isoforms), named for their relative sizes
2. rhGH has only one GH isoform (22kDa)
3. After injection of rhGH, the 22kDa isoform
dominates 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
rhGH Injection
One isoform only:
22kDA
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 doping
with 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
9
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:”Gene
therapy 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?
10
Degrees of potential genetic
interevention
• 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 Code
The 2008 Prohibited List
PROHIBITED
SUBSTANCES
• 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
SUBSTANCES
PROHIBITED IN
PARTICULAR SPORTS
• P1. Alcohol
• P2. Beta blockers
11
12
13
14
• 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); …
15
Sport authorities Concerns
• 2001: Gene Therapy Working Group,
convened by the IOC-MC meeting:”Gene
therapy 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 and
unrecognized pitfalls and dangers.
•
The participation of physicians in gene transfer procedures that are not fully
compliant with standards of human clinical research should be considered
medical malpractice.
•
Greater interactions should be encouraged to stimulate awareness of the
potential illicit use of gene transfer techniques .
•
New detection methods are likely to emerge and will help to prevent tainting of
sport by gene doping. Research programs should be supported.
•
The use of genetic information to select for or discriminate against athletes
should be strongly discouraged.
•
Sports organizations should promote knowledge about the potential dangers
associated with the misuse of genetic manipulations.
16
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 and
unrecognized pitfalls and dangers.
•
The participation of physicians in gene transfer procedures that are not fully
compliant with standards of human clinical research should be considered
medical malpractice.
•
Greater interactions should be encouraged to stimulate awareness of the
potential illicit use of gene transfer techniques .
•
New detection methods are likely to emerge and will help to prevent tainting of
sport by gene doping. Research programs should be supported.
•
The use of genetic information to select for or discriminate against athletes
should be strongly discouraged.
•
Sports organizations should promote knowledge about the potential dangers
associated with the misuse of genetic manipulations.
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 and
unrecognized pitfalls and dangers.
•
The participation of physicians in gene transfer procedures that are not fully
compliant with standards of human clinical research should be considered
medical malpractice.
•
Greater interactions should be encouraged to stimulate awareness of the
potential illicit use of gene transfer techniques .
•
New detection methods are likely to emerge and will help to prevent tainting of
sport by gene doping. Research programs should be supported.
•
The use of genetic information to select for or discriminate against athletes
should be strongly discouraged.
•
Sports organizations should promote knowledge about the potential dangers
associated with the misuse of genetic manipulations.
17
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 and
unrecognized pitfalls and dangers.
•
The participation of physicians in gene transfer procedures that are not fully
compliant with standards of human clinical research should be considered
medical malpractice.
•
Greater interactions should be encouraged to stimulate awareness of the
potential illicit use of gene transfer techniques .
•
New detection methods are likely to emerge and will help to prevent tainting of
sport by gene doping. Research programs should be supported.
•
The use of genetic information to select for or discriminate against athletes
should be strongly discouraged.
•
Sports organizations should promote knowledge about the potential dangers
associated with the misuse of genetic manipulations.
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 and
unrecognized pitfalls and dangers.
•
The participation of physicians in gene transfer procedures that are not fully
compliant with standards of human clinical research should be considered
medical malpractice.
•
Greater interactions should be encouraged to stimulate awareness of the
potential illicit use of gene transfer techniques .
•
New detection methods are likely to emerge and will help to prevent tainting of
sport by gene doping. Research programs should be supported.
•
The use of genetic information to select for or discriminate against athletes
should be strongly discouraged.
•
Sports organizations should promote knowledge about the potential dangers
associated with the misuse of genetic manipulations.
18
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 and
unrecognized pitfalls and dangers.
•
The participation of physicians in gene transfer procedures that are not fully
compliant with standards of human clinical research should be considered
medical malpractice.
•
Greater interactions should be encouraged to stimulate awareness of the
potential illicit use of gene transfer techniques .
•
New detection methods are likely to emerge and will help to prevent tainting of
sport by gene doping. Research programs should be supported.
•
The use of genetic information to select for or discriminate against athletes
should be strongly discouraged.
•
Sports organizations should promote knowledge about the potential dangers
associated with the misuse of genetic manipulations.
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 and
unrecognized pitfalls and dangers.
•
The participation of physicians in gene transfer procedures that are not fully
compliant with standards of human clinical research should be considered
medical malpractice.
•
Greater interactions should be encouraged to stimulate awareness of the
potential illicit use of gene transfer techniques .
•
New detection methods are likely to emerge and will help to prevent tainting of
sport by gene doping. Research programs should be supported.
•
The use of genetic information to select for or discriminate against athletes
should be strongly discouraged.
•
Sports organizations should promote knowledge about the potential dangers
associated with the misuse of genetic manipulations.
19
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)
Growth Hormone / IGF-1
Laboratory mice transferred with IGF-1 gene
at the University of Pennsylvania seem to hold
the “beneficial” effects of IGF-1 throughout
their lives.
20
Myostatin inhibition
In 1997, scientists
McPherron and Lee
revealed to the
public the ‘secret’
of an anomaly that
livestock breeders
have capitalized
since the late
1800’s: the gene
responsible for big
beefy cows .
Transgenic rats
A german boy had
mutation with lack of
myostatin production
(Schuelke M, N Eng J Med
2004, 350: 2682-2688)
Genes expressing rEPO
CMV
mEpo
SV40
Neo
Scheme of the mEpo vector
used in our animal gene
transfer studies
21
Awaiting for gene transfer
Anaesthesia
Plasmid injection
Tibialis muscle exposed
Applying a conductive gel
Setting electrical parameters
Muscle width measurement
Electroporation
Work done!
Effects after gene transfer to mice muscle
Serum EPO
14000
12000
Hematocrit
8000
Control
Transfected
6000
90
4000
85
2000
80
75
0
0
2
4
6
8
Time (days)
10
12
14
16
HCT%
[EPO] mU/ml
10000
70
65
60
55
50
2
8
14
Time (days)
22
Gene therapy with EPO
Monkeys injected with a
virus carrying the gene
for EPO
23
Possible strategies for detection
gene 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 detection
gene 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
24
IMAGENE
Non 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 one
Imagine, John Lennon, 1974
RESEARCH HYPOTHESIS AND OBJECTIVES
AAA
- The gene transfer processes may
produce the expression of mRNA for the
target hormone-protein in unusual cells
or tissues.
AAA
Cap
Cap
- mRNA molecules will hybridize with
suitable
antisense
modified
oligonucleotides available to the tissue
expressing the ectopic hormoneprotein.
- 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.
Imaging
Labeled TAT-PNA preparation
Anaesthesia
Administration
Quality control
Preparing administration I
Image adquisition I
Chromatograms
Preparing administration II
Image adquisition II
25
26
Final Aim : Sensitivity and Resolution of Positron
Emission Tomography (PET) or Single Photon Emission
Computerized Tomography (SPECT)
PET
facility
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.
27
28