GH
• ”The most expensive, most fashionable and least understood of the new athletic drugs.”
(Underground Steroid Handbook 1983)
Could be – but does it work?
Clinical Endocrinology (2009) 71, 860–866
Isometric muscle strength
Mata Analysis of Placebo-Controlled Trials
Clinical Endocrinology (2009) 71, 860–866
Impact of GH substitution on exercise capacity and muscle strength in GH-deficient adults:
a meta-analysis of blinded, placebo controlled trials
Aerobic exercise capacity
Ann Intern Med. 2010;152(9):568-577 • Placebo-controlled,
double blind GH trial (2 mg/d) in 96 recreationally trained athletes (63 men and 33 women) for 8 weeks + 6 weeks washout
• Measurements included body composition, muscle strength, VO2-max, sprint capacity (wingate value)
• Supported by WADA
17
Challenges in GH abuse detection
• rhGH is almost identical to pituitary GH
• GH is secreted in a pulsatile manner
– Exercise & stress increase GH secretion
• Urine testing is not feasible • The presence of a prohibited substance or its
metabolites or markers in an athlete’s bodily specimen’’…constitutes an offence (WADA).
Hartman et al, J Clin Endocrinol Metab 1992; 74: 757
GH secretion is pulsatile and episodic!
24 h profile of serum GH concentration and deconvolution-resolved GH secretory rate
• Narrow window of opportunity (< 24 h) • Detects only biosynthetic 22k doping • Does not detect GH secretagogs
”Isoform method”
GH 2000 • UK, Sweden, Italy, Denmark Joint Study
supported by EU-IOC (1997-2000) • Protocols:
– High dose GH in healthy subjects – GH and related markers in athletes
• Cross-sectional • Prospective • Before, during and after competition
• Results: – 20 peer-review publications – A method…
Discriminant analysis: The combination of IGF-I and P-III-P Provided the best model Corrections for age and gender A pre-defined specificity of 1:10000 Sensitivity: Males: 86% Females: 60%
GH & IGF Res 2007;17:220-226
25
Treatment 0.06 IU/kg/day GH Placebo
Detection of dopers with GH-2000 formula
GH
tre
atm
ent
6 . 0 4 . 8 3 . 7 2 . 4 1 . 2 0 . 0 - 1 . 2
0 3
5 7 9
11 13 16
19
Visit day of study
GH-2000 formula score
“Non-doped” “Doped”
Overall sensitivity
90%
Erotokritou-Mulligan GH& IGF Research 2007 17(5):416-23. Epub 2007 Jun 20
IOC Rome Workshop 1999
• It is possible to detect exogenous GH administration for up to 2 weeks after the last dose
– Principle was scientifically sound
– Sensitivity and specificity reasonable
• Blood testing and immunoassays will be necessary
• Results obtained were in a predominantly white European population
• More data on the effect of injury needed
…and so the GH-2004 project was established
Ethnicity 1200
1000
800
600
400
200
0
10 20 30 40 50 60 70
Age (years)
IGF-I (ng/ml)
Caucasian (n=493) Oriental (n=25) Afro-Caribbean (n=191) Indo-Asian (n=19)
* Other (n=29)
Injury
Number of Days since Injury
2
0
-2
-4
7±3
14±3
21±3
84±7 28±3 0-3
42±7
GH-2000 score
Men (n=127)
Further work: Adolescents
• GH-2000 score increases during puberty
• No false positives
Current state according to the inventor • The Biomarker is formally approved by WADA/IOC at
the London Olympic Games and caught two para-olympians who were missed by the iso-form test. Both admitted doping and went home.
• The test is currently in limbo as there are commercial assay problems…
• Maybe measurement of IGF-I and PIIINP with MS may solve the problem?
Jorgensen
Excuse me…I know the game is over, but just for the record, I don’t think my buzzer was
working properly
Validation in other populations/ labs
• Test worked in other studies
– Germany
– Australia
– Rome
34
Determining Decision limits to be used in anti-doping
• New limits with currently available assays that should allow introduction of test for anti-doping purposes
35
39 High dose GH
Change in IGF-I and P-III-P with GH
0
0.5
1.0
1.5
2.0
2.5
3.0 P-III-P
Pre GH On GH 0
500
1000
1500
2000
2500
3000
Pre GH On GH
IGF-I
Low dose GH Placebo
P-III-P (U/ml)
IGF-I (ng/ml)
Eur J Endocrinol. 2010 Jul;163(1):45-54
40
Effect of Exercise on IGF-I
0
10
20
30
40
50
60
70
80
90
0 15 30 45 60 75 90 105 120 135 150
GH
Placebo
IGF-
I
Time (minutes)
Exercise
Wallace et al JCEM 1999. 84: 3591-601
41
Institut für Dopinganalytik und Sportbiochemie Kreischa GH Study
• 15 healthy male, non-competitive athletes • Age 21–33 yrs, mean 24 years • BMI 19.4–31.7 kg/m2
• Two treatment groups – placebo (n=5) – rhGH 0.06 IU/kg/day (n=10)
• 14 days therapy by injection • Blood samples taken prior to, during and until 4 weeks after
treatment.
Kniess et al Anal Bioanal Chem. 2003 Jul;376(5):696-700
42
Response of GH-dependent markers in an ethnically diverse
group
• 31 male & 13 female healthy volunteers recruited from gyms & University of Southampton sports clubs
• 19 Afro-Caribbean, 21 Indo-Asian, 4 Oriental
• Randomly assigned to three treatment groups: – Placebo
43
Effect of Injury
• No significant change in IGF-I
• 40-70% rise in P-III-P – Dependent on type and severity of injury
2
0
-2
-4
7±3
14±3
21±3
84±7 28±3 0-3
42±7
Number of Days since Injury
GH-2000 score
Men (n=127)
2
0
-2
-4
Women (n=40)
7±3
14±3
21±3
84±7 28±3 0-3
42±7
Erotokritou-Mulligan et al (2008) J Clin Endocrinol & Metab 93(7):2760-3
Fat
STAT5b is the major signaling protein
GH + − − + 60
4 + − 30
2 3 − +
30
1
+ − 60
5
+ − 30
6
60
Subject #
p
t
p
t
Muscle
Fat
AU
0,00
0,05
0,10
0,15
0,20
0,25
Muscle Saline GH Saline GH
P=0.001 P=0.07
Am J Physiol 2006;291:E899-E905
GHR-GH-GHR
JAK2
STAT5b
SOCS IGF-I
PI3-kinase PKB/Akt
MAPK ERK1/2
Src
JNK
GH signalling pathways documented in vivo in muscle and fat in human subjects
AJP 2006;291:E899-905 JCEM 2008;93:2842-50
Time (minutes)-50 0 50 100 150 200
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
GHSaline
0
20
40
60
80
100
120
(µg/l)
GH
FFA (mmol/l) P = 0.03
Am J Physiol 2006;291:E899-E905
Identified n = 137
Retrieved n = 49
Excluded n = 88
Excluded n = 23
Fulfilled Inclusion n = 26
New Articles n = 4
Non-Extractable
data n = 13
Meta-analysis n = 17
Effect of GH substitution on exercise capacity and muscle strength: a meta-analysis
Clinical Endocrinology (2009) 71, 860–866
Jorgensen 1996
Vahl
Wallymahmed
Whitehead
Jørgensen 1989
Total
Muscle Strength n = 113
weighted mean ± SE: 2,8 ± 2,2 % (p =0,20)
Juul et al. JCEM 1994, 78: 744-752
IGF-I is an important mediator of the anabolic GH effects (but serum IGF-I mainly reflects hepatic production)