Drug Testing

1
Drug Testing

• William W.Dexter, MD., FACSM
• Maine Medical Center
• Sports Medicine Program

2
Is the (sporting) Apocalypse upon us?

• Chinese system in place for 2008 . . . .
• 185,000 athletes ages 7-11 at 1780 sports schools
• 56,000 secondary school age at 2100 sport
  colleges
• 20,000 elite at 195 centers

3
Is the (sporting) Apocalypse upon us?

• Chinese system in place for 2008 . . . .
• Earning gold the old fashioned way?
• thousands of sports scientists . . . .

4
Meanwhile back in the USA..
Big leaguers linked to steroids, HGH
5
Goals

• History
• Rationale
• Components
• Procedures
• WADA
• Controversies

6
History

• 1960s IOC defines doping, creates list
• 1970s IOC begins formal testing
• 1980s NCAA creates guidelines
• 1990s Anabolic Steroids Control Act
• 1995 US Supreme Court - HS testing

7
History

• 2000-1 new r-EPO test
• Cycling
• 2003 Genetic Doping added to list IOC
• 2004 Anabolic Steroid Control Act

8
Drug Testing Programs
Education in and of itself never changed human
behavior Robert Voy, MD . . . Ethical
considerations . . Not effective USOC Detect,
Deter, Level field

• Why
• clear purpose

9
Drug Testing Programs

• Who
• some or all
• voluntary v. mandatory
• What
• performance enhancers
• street drugs

10
Drug Testing Programs

• When
• random best
• competition v. training
• How
• to test
• to sanction
• to pay for it

11
Protocols

• IOC/USOC
• NCAA
• MLB
• NBA
• NFL
• HS

12
Methodology

• Urine (v.blood)
• Chain of custody
• Certified lab
• Substance
• metabolites
• High lower limit

13
Collection and Chain-of-Custody Issues

• Three facets of the testing process
• collection
• transportation
• laboratory analysis
• Typical protocol sample to be divided into A
  and B bottles
• WADA rigorous certification process for labs
  that to perform testing.
• 2 in North America (Montreal and Los Angeles).

14
Technique

• The differentiation of peptide or protein drugs
  from endogenously produced compounds
• key factor in sports drug testing
• characteristic differences
• molecular masses
• modified amino acid sequences
• results in different chromatographic,
  electrophoretic, mass spectrometric behavior
• mandatory in reports of adverse findings.

15
Technique

• Tests
• Immunoassays first
• GCMS confirmatory
• Carbon isotope ratio testing (IRMS)
• Differentiate exogenous naturally occurring
  testosterone
• Testing errors
• false positives
• human
• machine

16
Mass spectrometry essential tool in doping
control analysis

• Gas chromatographycombustionIRMS
• determine if T/E ratio is result of exogenous
  testosterone
• detect small differences in isotopic composition
• international reference standard
• based on
• 98.9 of carbon atoms in nature are 12C
• 1.1 13C (isotope of carbon contains an
  additional neutron)
• Liquid chromatography
• Chromatographic separation of peptides and
  proteins
• high-performance liquid chromatography (HPLC)
• robust and reproducible

Thevis, et alRapid Commun. Mass Spectrom. 2007
21 297304
17
Technique

• Identified over 5000 compounds
• Allow tree for classification
• Simplify process
• allow detection
• protect athletes

18
Testing and Detectability(From Green, AJSM, 2006)
19
Interpretation of Drug-Testing Results

• Two types of drug-testing results
• (1) the presence of substances for which any
• amount represents a positive test result
• (2) tests that have cutoffs or thresholds of
  reporting
• imperative to understand the limitations of a
  positive test result.

20
Substances With Threshold Reporting
Levels Substance Reporting Threshold Caffeine
15 µg/mL (a) Cathine 5 µg/mL Ephedrine
1.5-10 µg/mL (b) Epitestosterone 200
ng/mL Methylephedrine 10 µg/mL Morphine 1
µg/mL Norandrosterone 2 ng/mL,
males Salbutamol 1 µg/mL THC 15
ng/mL Testosterone/ epitestosterone gt41 gt61
(c) A Caffeine is monitored by the WADA B WADA
and NCAA threshold is 10 µg/mL NFL is 1.5
µg/mL. C WADA and NFL threshold is 41 the NCAA
threshold is 61.
(From Green, AJSM, 2006)
21
Sanctions

• Depends on
• sport
• level
• substance
• Range
• warning
• treatment
• expulsion

22
Anabolic Steroids

• Banned by all
• Felony to provide
• Testing
• substance or metabolite

23
Testing

• Steroids
• TE lt/ 6
• Methods exist to keep TE normal
• New test CIR
• Ratio of carbon -13 isotope carbon-12
• Synthetics have less C-13
• Time course studies

24
TE Ratio

• Old standard TE ratio (gt61)
• subvert test
• take epitestosterone
• Epitestosterone now quantified
• gt 200 ng/mL positive
• WADA lowered the threshold in 2005
• T/E ratio of gt 41 requires further
  investigation.
• Rare natural occurrence
• Repeat measures recommended

25
Exogenous (xenobiotic) v Endogenous

• GC-MS can be operated in either the
• Full scan mode
• Ion-monitoring mode (More sensitive).
• Steroid-screening tests are conducted in ion
  monitoring
• If a banned drug is detected
• Confirmation via full-scan mode
• High-resolution MS
• Extends the period of detectability of exogenous
  AAS.

26
Concern over contaminants
sIRMS provides conclusive evidence of the origin,
endogenous or exogenous, of the urinary
metabolites, even when found in low
quantities. Ayotte, Br. J. Sports Med.
20064025-29
27
Special case nandrolone (19NA)

• Small amounts can occur without ingesting
• Pregnant women excrete small amounts
• Oral contraceptives containing norethisterone
• Some males may naturally produce miniscule
  amounts
• Therefore
• 19 NA reported with a specific concentration
  level. 2 ng/mL
• OCP resolved by the finding of specific
  norethisterone metabolites.
• Injectable nandrolone decanoate
• detected by urine testing for months to a year
• ???? repeat positive test result represents
  continued use.

Ayotte C.Br J Sports Med. 2006
28
Stimulants

• Amphetamines
• prevalence on rise
• banned IOC, NCAA
• easily detected
• Cocaine
• commonly abused
• prevalence declining
• banned IOC, NCAA
• easily detected

29
Stimulants

• Sympathomimetics
• ephedrine pseudoephedrine phenylpropanolamine
• common use, ?effective
• Caffeine
• NCAA bans
• WADA allows

30
Stimulants

• Beta-2 Agonists
• inhaled allowed
• clenbuterol - banned
• Caffeine
• CNS at 100 mg
• ergogenic at 300 mg
• lethal at gt3gms
• 6 real cups (dq)
• Nicotine

31
Detection

• The method consists of a liquid-liquid
  extraction with diethyl ether at pH 14 and
  analysis of the extracts with a LCQ-Deca1 mass
  spectrometer equipped with an atmospheric
  pressure chemical ionisation interface, operated
  in positive ionisation mode.
• Deventer,etal, Rapid Commun. Mass Spectrom. 2006

32
Other Substances

• Beta Blockers
• ergogenic v. ergolytic
• testing varies
• Alcohol
• banned shooting
• HSs some test
• Marijuana
• IOC may be tested
• NCAA banned

33
Blood Doping (Green, AMSSM)

• Altitude training - allowed
• Artificial Altitude allowed (but opposed?)
• Blood transfusion - banned
• rEPO/EPO - banned
• Darbepoetin(Arnesp) - banned
• Repoxygen - banned
• HES - banned
• RSR-13 not banned

34
Blood doping recent Hx

• EPO
• 1998 Tour de France Festina masseur caught
• Festina ejected, 7/9 admit doping
• Marco Pantani won
• ejected Italian race 1999
• Dies 2004 , HCT reported 60
• Russian, US runners
• Kelli White
• Darbepoetin
• SLC Olympics
• 3 skiers, 9 medals
• Darbepoetin on market 3 months
• Hydroxyethyl starch (HES)
• Masks EPO
• Finnish, German athletes

35
Blood Doping

• Blood tests index of markers
• hematocrit, reticulocytes, and iron parameters,
  OFF score
• Individual reference values
• Algorithm (Hgb)
• No set method

36

• WADA
• Quantify antigenically distinct rbcs
• Flow cytometry
• Can identify with lt5

37
Homologous Transfusion

• Figure 3 Anti-doping blood analysis demonstrating
  the presence of antigens
• Measuring red blood cell proteins

Robinson Br. J. Sports Med. 2006
38
Autologous Transfusions

• Undetectable (for the time being)
• Policy of No Start
• Introduced by some federations
• Will strongly limit this doping strategy
• Based on individual reference values
• Each person own set of reference values
• Variations and evolution of the markers over time
• Announced and unannounced

39
R-EPO detection

• r-HuEPO (1987), darbepoetin (2001)
• nearly identical to natural EPO
• detection by conventional means impossible.
• Urine method
• direct test for r-HuEPO
• isoform patterns of EPO
• distinctively different from natural EPO. test
  also yields a pattern for
• Darbepoetin is distinct from EPO and r-EPO

From Green AJSM 2006
40
Where there is a will

• There is a way..
• Sub clinical r-epo
• Increases rbc,
• Draw off 3-5 l/month
• Store
• Use in competition, avoid detection
• Autologous transfusion

41
Blood Doping New and Improved

• Epoetin delta (Dynepo)
• Produced in human cell lines hard to detect
• RSR-13
• 2,3 DPG mimetic
• o2 curve shifts right
• Increase Vo2max
• Oxygen transport substitutes
• perfluorocarbons

42
Concerns testing

• ? Training effects on r-epo
• No training effect
• Exception
• recreational athletes, 21 km race
• Tourniquet time
• May have some effect on parameters

Abellan, IntJSportsMed 2007
Lippi, IntJSportsMed 2006
43
Emerging Issues
44
Selective Androgen Receptor Modulators (SARM)

• Full agonist in anabolic targets(muscle)
• Partial or non agonist in others (prostate)
• Huge potential for abuse
• 4 classes
• (1) aryl-propionamide
• (2) bicyclic hydantoin
• (3) quinoline
• (4) tetrahydroquinoline

Thevis, etalRapid Commun. Mass Spectrom. 2006
45
Selective Androgen Receptor Modulators (SARM)

• preventive anti-doping research
• Four model SARMs synthesized and used to
  establish a fast, sensitive and robust screening
  assay using LC/MS/MS.

Thevis, etalRapid Commun. Mass Spectrom. 2006
46
Hypobaric
47
Hypobaric

• More effective than altitude
• Does this violate spirit?
• Wada considered for 2007 banned list
• Levine (and others) argue against
• Ex saunas, living at altitude

Levine, B Scand JMedSciSports 2006
48
Peptides

• hGH
• Banned
• Undetectable
• Parenteral administration
• 20 minute half-life
• peaks in 1 to 3 hours
• imperceptible at 24 hours

Saugy Br J Sports Med 2006
49
Peptides

• Direct method
• 2004,6 Olympics
• None finally declared
• Time window too-short
• Indirect
• Markers of hGH use
• IGF-1
• Procollagen type III
• Consistently discriminates

Saugy Br J Sports Med 2006
50
Peptides

• hCG, LH, GnRH
• no assays detect
• Insulin like growth factor (IGF-1)
• Vascular endothelial growth factor (VEGF)
• Myostatin
• Endorphins, Enkephalins

51
Emerging Issues Genetic Doping

• Defined
• non therapeutic use of genes, genetic elements
  and/or cells that have the capacity to enhance
• performance
• Included in prohibited classes

52
Emerging Issues Genetic Doping

• History
• 1964 Mantyranta
• Genetic mutation for RBC production with o2
  tension
• Insert gene via adenovirus
• Repoxygen
• Muscle wasting disease
• IGF1, MGF

53
Genetic Doping

• Complicated, costly
• Detection
• Difficult ? Impossible
• No urine/blood
• Focal applications (muscle)
• ? Measure effects
• barcodes

Haisma, IntlJSportsMed, 2007
54
Testing

• New directions
• Timing hold samples and test at later date
• Retroactive indemnification
• NCAA
• Testing authorized
• All Div I sports
• Random year round
• Local in house

55
Hair testing

• Drug incorporates in
• Sweat, sebum
• Varies with serum
• Grows .44mm/day
• Collection easier than urine
• Hair analysis methods
• Immunological
• Gas/liquid chromatography
• Capillary electrophoresis.
• DNA confirms, second sample easy
• External contamination
• Ex cosmetics

Kintz,Ther Drug Monit. 2006.
56
Hair testing

• Hair is not yet approved by the International
  Olympic Committee (IOC),
• It is accepted in most courts of justice that
  have dealt with such cases

Boumba, etal, International Journal of
Toxicology, 2006
57
World Anti Doping Agency(WADA)

• World Anti Doping Code
• Harmonize regulations
• Clearing house
• Passport system
• Web based, hard copy
• 2 way system
• Confidential
• ADAMS
• Anti doping administration system

58
World Anti Doping Code

• International
• Testing Standards
• Laboratory standards
• TUE
• Application in 21 days prior
• Significant impairment
• No alternative
• No enhancement
• Not retroactive
• (unless extraordinary)

59
World Anti Doping Code

• STRICT liability for test
• Prohibited substances list
• Tentative Provisions
• Consistent sanctions
• Allow exceptional circumstances
• Reduced penalties for those who cooperate

• Research priorities
• Steroids, Blood doping, HGH

60
WADA 2004

• Removed from list
• Decongestants
• Caffeine
• Added to list
• Modafinil
• Beta blockers specifics
• Etoh limits set
• Salbutamol
• Urine concentration set
• Can get TUE

61
WADA 2007Prohibited ListSummary of Major
Modifications

• S9. Glucocorticosteroids
• examples of glucocorticosteroid routes of
  administration added to provide better guidance
  to TUE
• P1. Alcohol
• At the request of the WCBS, Billiards is
  removed from this section.
• P2. Beta-Blockers
• At the request of the FIDE, Chess is removed
  from this section.
• Other minor changes

62
World Anti Doping Code

• Cooperation and the Code
• Recent problems
• Tour de France
• USATF/USOC
• Contributions lagging

63
Awareness, Attitudes and Administration

• WADA v
• National agencies
• Sports federations
• Pace of change

64
Awareness, Attitudes and Administration

• Credibility
• MLB
• Star system
• Behind the scenes
• Pharmaceutical industry
• Clenbuterol and Krabbe
• ?WADA sponsor
• Pro v amateur

65
Awareness, Attitudes and Administration

• Confused on what should be done?
• So are they
• Optimal policy equation

Barro-Gordon type approach to doping policy (Eber
2002)
66
Athlete v. The System

• Many athletes live in an atmosphere of
  exemption from the rules (Will)
• if (they) thought it would help theyd take
  rat manure (Ziegler)

67
Athlete v. The System

• Drug use in elite sports is epidemic drug
  testing is a farce
• Yesalis
• Ignorance is never an excuse (for not beating
  the system)

68
Barriers to Effective System

• Cost
• time
• resource
• money (10-200/test)
• Lack of consensus
• methods
• substances
• sanctions
• Mixed message
• athletes
• society

69
Beating the System

• Operación Puerto
• Huge pan European doping ring
• Manipulate the sample
• chemically
• physically
• Careful use
• Legal challenges

70
Is there an effective system?

• Olympics
• 2/3s polled not effective deterrent
• data conflicting
• high prevalence known
• low incidence shown
• Elite athlete would you use if . . .?

71
Is there an effective system?

• NCAA
• Banned list
• Most street ergogenic
• Rule 1.3
• wide range
• substances
• sanctions

72
Is there an effective system?

• High Schools
• Varies by state/school
• Legal challenges
• Mostly street drugs
• More useful than NCAA and IOC ?
• Data conflicting

73
Should there be any system?

• If (athletes) continue to use drugs, then we, as
  physicians, must provide monitoring and advice on
  dosing and drug free times to ensure that the
  risks are minimized
• editorial, BJSM
• . . .Id show them the door straight away, with
  a swift kick up the ass . . .!
• John Treacy, Silver medalist

74
Doping Control for the Team Physician A Review
of Drug Testing Procedures in Sport

• 2005 NCAA Survey of Use and Abuse Habits of
  Collegiate Athletes.
• When anabolic steroid users were asked to
  identify their sources of the drugs, 13 named a
  physician, which was a leading response
• Green Am. J. Sports Med. 2006 34