Search for additional biomarkers – multi-layered system

1Progress report – 06.11.19

Human genome

Transcriptome

Proteome

Metabolome

Human systems

Influenced Biomarkers

Exposome

Hypoxia

Steroids

Exercise

Blood doping

Gender

Transcriptome

Transcriptomic biomarkers of blood doping: autologous blood transfusion

2Progress report – 06.11.19

Blood Donation: ~500mL Blood Transfusion

Transfusion Clinical study 36 days of storage at 4°C

Tempus RNA blood tubes

+3h-1 +1 +3+6h-4 +6+12h +2 +9 +15

Blood manipulation

Bone marrow

Erythropoiesis

Blood circulation Immature red blood cell

Red blood cell

Maturation

mRNA

Protein

EDTA tubes

Confirmation using RT-qPCR and correlation with IRF

3Progress report – 06.11.19

ALAS2CA1SLC4A1

00.5

11.5

2

-4 -2 0 2 4 6 8 10 12 14 16

RETI

CULO

CYTE

S %

Days

RET%

Outcomes 1

4Progress report – 06.11.19

20172016

Blood transcriptome impacted by autologous blood transfusion

ALAS2, CA1, SLC4A1 genes as principal candidate markers

Variations more important than for conventional hematological parameters

Longitudinal monitoring of the number of transcripts may provide additionalevidence of blood doping

Dried blood spots for antidoping purpose

5Progress report – 06.11.19

Minimal invasiveness

Easy sampling

Increased frequency of sample collection

Cost-effective transport

Direct detection of doping drugs

Quantification of biomarkers

Quantification of ALAS2 RNA expression in dried blood spots

6Progress report – 06.11.19

DBS excision

QIAzolSonication

Incubation

Chloroform Centrifugation Transfer of

aqueous phase Elution

miRNeasy Mini kit

ALAS2 LinearALAS2 Linear + Circular

Amplification Data treatmennt

2. RT-qPCR

1. RNA extraction

ALAS2 gene expression after blood withdrawal

7Progress report – 06.11.19

0

0.5

1

1.5

2

2.5

-1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

Retic

uloc

yte

%

Time (days)

RET%

0

2

4

6

8

10

12

14

16

18

-1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

Imm

atur

e re

ticul

ocyt

e fr

actio

n (%

)

Time (days)

IRF%

**

-1 3 6 9 15 30210 (days)

TempusEDTADBS

Blood donation450 mL 16 healthy male volunteers

ALAS2 gene expression after blood withdrawal

8Progress report – 06.11.19

0

100

200

300

400

500

600

700

-1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

ALAS

2/Re

f

Time (day)

Tempus® Tube

ALAS2L

ALAS2L+C

*** ** **

*

** *****

0

100

200

300

400

500

600

700

800

-1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

ALAS

2/Re

fTime (day)

Dried blood spot

ALAS2L

ALAS2L+C

*

*** ****

-1 3 6 9 15 30210 (days)

TempusEDTADBS

Blood donation450 mL 16 healthy male volunteers

ALAS2 gene expression after rhEPO injections

9Progress report – 06.11.19

0

20

40

60

80

100

120

-3 -2 -1 0 1 2 3 4 5 6 7 8

ALAS

2/Re

f

Time (day)

Volunteer 2

ALAS2L+C

ALAS2L

0

20

40

60

80

100

120

-3 -2 -1 0 1 2 3 4 5 6 7 8

ALAS

2/Re

f

Time (day)

Volunteer 1

ALAS2L+C

ALAS2L

-3 3 4 7210 (days)

DBS

2 healthy male volunteers= 3’500 IU Eprex

Outcomes 2

10Progress report – 06.11.19

DBS is a suitable matrix for gene expression analysis

Expression of ALAS2 gene is stimulated after blood withdrawal and rhEPO injections

Inclusion of ALAS2 circular form improves the magnitude of the response observed

Confirmation that ALAS2 is a promising candidate for the detection of blood doping through longitudinal monitoring

No difference between venous and capillary blood for quantification of ALAS2transcripts

Biomarkers of testosterone doping - steroidomics

11Progress report – 06.11.19

Human genome

Transcriptome

Proteome

Metabolome

Human systems

Influenced Biomarkers

Exposome

Hypoxia

Steroids

Exercise

Blood doping

Gender

Metabolome

Extended profiling of serum endogenous steroid

12Progress report – 06.11.19

Loading 200 µL serum + 200µL H3PO4 4%Washing 400 µL H2O/MeOH 95/5 (v/v) + 0.1% NH4OHElution 50 µL MeOH/H2O 90/10 (v/v)Dilution 50 µl H2O

SPEOASIS HLB µElution (Waters)

Kinetex C18 150 x 2,1 mm , ID 1.7 µm

Flow rate 300 µL/min Temperature 55°C

Phase A H2O + 5mM Ammonium Formate

Phase B MeOH + 5mM Ammonium Formate

Gradient 40% B to 80% B in 20 min

Sample preparation

UHPLC-MS/MS analysis

11-Deoxycorticosterone11-Deoxycortisol17a-Hydroxyprogesterone21-Deoxycortisol5ααβ-Adiol (5α-diol)5αββ-Adiol5βαβ-Adiol (5β-diol)5βββ-Adiol

AndrostenedioneAndrosteroneCorticosteroneCortisolDHEADHTEpitestosteroneEtiocholanolone

ProgesteroneTestosterone

5ααβ-Adiol, 17-Gluc5ααβ-Adiol, 3-Gluc5αββ-Adiol, 3-Gluc5βαβ-Adiol, 17-Gluc5βαβ-Adiol, 3-GlucAndrosterone-3-GlucDHEA-3-GlucDHT-17-GlucEpiAndrosterone-3-GlucEpitestosterone-17-GlucEtiocholanolone-3-GlucNorAndrosterone-3-GlucNorEtiocholanolone-3-GlucTestosterone-17-Gluc11-Ketoetiocholanolone-GlucPregnanediol-3-Glu

11b-Hydroxyandrostenedione11b-Hydroxyandrosterone11b-Hydroxyepiandrosterone11-Dehydrocorticosterone11-Ketoetiocholanolone16a-Hydroxyandrostenedione 16a-Hydroxyprogesterone16a-Hydroxytestosterone16-Androstenol18-Hydroxycorticosterone20a-Cortolone20a-Dihydrocortisone20b-Cortolone3a,5a-Tetrahydrocortisol3a,5b-Tetrahydrocortisol3a,5b-Tetrahydrocortisone7a-HydroxytestosteroneAdrenosteroneCortisonePregnanediolPregnenolone

Androsterone-3-SulfDHEA-3-SulfDHT-17-SulfEpitestosterone-17-SulfEtiocholanolone-3-SulfNorEtiocholanolone-SulfTestosterone-17-SulfEpiandrosterone-3-SulfDehydroandrosterone-Sulf5abb-diol-17-Sulf11-Ketoetiocholanolone-Sulf

Free steroids and Phase I metabolites Phase II metabolites

TestoFem Clinical study

13Progress report – 06.11.19

Authorized by

• 14 healthy women• No hormonal contraception• Regular menstrual cycles• No high-level sport

Study design: 3 phases = 3 menstrual cycles

Phase 1: Pre-treatment monitoring

Phase 2: Testosterone gel treatment

Phase 3: Post-treatment monitoring

Funded by

TestoFem Clinical study

14Progress report – 06.11.19

So far

• 12 volunteers screened

• 7 volunteers included

• 2 volunteers completed the study

• 1 volunteers completed treatment phase (phase III on-going)

• 4 volunteers under treatment (phase II)

• No adverse effect linked to the product reported

• No drop-out

• No treatment effect on the menstrual cycle

TestoFem Clinical study – Preliminary results steroid profile UHPLC-MS/MS

15Progress report – 06.11.19

Testosterone

Jours

Testo

stero

ne (n

mol/

L)

12

34

5

J+1 J+17 J+23 J+29 J+32 J+40 J+50 J+54 J+60 J+71 J+79 J+82

DHT

Jours

DHT

(nm

ol/L

)

0.5

1.0

1.5

2.0

2.5

J+1 J+17 J+23 J+29 J+32 J+40 J+50 J+54 J+60 J+71 J+79 J+82

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Test

oste

rone

(nm

ol/L

)

Jours

Testosterone

V1

V2

V3

V4

V5

V6

V7

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

0 10 20 30 40 50 60 70 80 90

DHT

(nm

ol/L

)

Jours

DHT

V1

V2

V3

V4

V5

V6

V7

Volunteer 1

Volunteer 2

Individual monitoring

Outcomes of the clinical study

16Progress report – 06.11.19

• Serum endogenous steroid profile (UHPLC-MS/MS analyses)

• Urinary steroid profile (GC-MS analyses)

• ‘Endocrinological’ profile - LH, FSH, SHBG, AMH, Ferritine, EPO (Immunology analyses)

• Hematological profile (Sysmex XN-1000)

• Targeted confirmatory GC-C-IRMS analyses

• Untargeted steroidomic study for the discovery of potential biomarkers

• Genotyping of UGT2B15 and UGT2B17 polymorphism

• Body composition (DEXA scans)

• Self-esteem (Rosenberg scale) and Quality of life (SF-36)

• Transcriptomic study (Tempus tubes and/or DBS)

• Evaluation of DBS as matrix for endogenous steroid profiling using UHPLC-MS/MS

Application of extended blood steroid profile to hyperandrogenic populations

17Progress report – 06.11.19

CAIS Neg PCOS

1000

2000

3000

4000

Population

X11bOHAdione

25

CAIS Neg PCOS

0100

200

300

400

Population

X16aOHTesto

DHT

11b-OH-Androstenedione 16a-OH-Testosterone

CAIS Neg PCOS

0100

200

300

400

500

Population

DHT 3646

50

CAIS Neg PCOS

02000

4000

6000

8000

Population

Testo

257

Testosterone

CAIS Neg PCOS

020000

40000

60000

80000

100000

140000

Population

AndroGluc

5

25

39

CAIS Neg PCOS

010000

20000

30000

40000

50000

60000

Population

EtioGluc

524

25

28

Androsterone Gluc Etiocholanolone Gluc

CAIS Neg PCOS

01000000

2000000

3000000

4000000

5000000

Population

AndroSulf

5

50

CAIS Neg PCOS

0100000

200000

300000

400000

500000

Population

EtioSulf

1

5

50

Androsterone Sulf Etiocholanolone Sulf

Differentiation of populations with steroid profile

Perspectives

18Progress report – 06.11.19

Inclusion of the 7 last volunteers for TestoFem clinical study

Preliminary analyses of the samples collected during the clinical trial

Validation of the quantitative UHPLC-MS/MS method

Comparison of the blood steroid profile between hyperandrogenicpopulations and doped volunteers

Development of a workflow for the UHPLC-MS/MS analysis of DBS

Comparison between urinary and blood steroid profile