Biomarkers in the pharmaceutical industry.
Translating research into clinical benefits
Peter Groenen, Sr. Director, Sr. Group leader Translational Biomarkers
COST CliniMARK– Summer School – 24 September 2019
Idorsia Pharmaceuticals Ltd.Founded June 16, 2017 as a demerger from Actelion-JnJ
COST CliniMARK Summerschool | 24 Sep 20192
50 minutes of roller coaster
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Everything presented and explained here today are my own words, thoughts and opinions. They do not represent those of Idorsia or any other organization I work or have worked for and are properly cited when derived
from a third party.
Also
My discussion may include predictions, estimates or other information that might be considered forward-looking. While these forward-looking statements represent my
current judgment on what the future holds, they are subject to risks and uncertainties that could cause actual results to differ materially. You are cautioned not to place undue reliance on these forward-looking statements, which reflect our opinions only as of the
date of this presentation. Please keep in mind that I am not obligating myself to revise or publicly release the results of any revision to these forward-looking statements in light of
new information or future events.
Disclaimer:
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The history of medicine and pharmaceutics in 5 minutes
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“IT'S FAR MORE IMPORTANT TO KNOW WHAT
PERSON THE DISEASE HAS THAN WHAT DISEASE
THE PERSON HAS.”
HIPPOCRATES
470-360 BC
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History of the pharmaceutical industry in 3 steps
- Until 1847 empirical research in humans
- Herbs, plants and minerals
- Since1847 Pharmacology as a scientific discipline
- Since1859 Chemical synthesis
- Empirical research in humans and animals
- Since the 1970’s: - Molecular biology- Experimental
research- Industrialization of
R&D: targeted rationalized drug discovery and development
The first generally
accepted use of plants as
healing agents was
depicted in the cave
paintings discovered in
the Lascaux caves in
France, which have been
radiocarbon-dated to
between 13,000 and
25,000 BC.
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Interim QuestionWhich Drug has saved most lives until now?
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A lot of drugs work very well fortunately
A life saver
Penicillin – 1942
Penicillin was first identified in 1928 (Fleming), but started to be used in 1942. (First isolation and in vivo efficacy demonstrated in 39 by Florey & Chain, first patient treated in 42). Turning point in human history and led the way in the treatment of numerous bacterial diseases.
It has been calculated that the antibiotic has saved over 80 million lives and without its discovery and implementation, 75% of people today would not be alive because their ancestors would have succumbed to infection.
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Interim QuestionWhich Drug was the biggest seller ever?
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Best selling drugs to date (in 2017)
Humira is probably going to take the 1st spot in 2020
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The modern pharmaceutical R&D
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Numbers
Current Pharmaceutical R&D
Target Discovery / Selection
Lead finding
Lead optimizatio
n
Pre-clinical developme
nt
Clinical Phase 1
Clinical Phase 2
Clinical Phase 3
Filing and registration
Post marketing activities
50-500 5-50 10-100 50-500
100-30000
100-1.109
Few years
after launch
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Target Discovery / Selection
Lead finding
Lead optimizatio
n
Pre-clinical development
Clinical Phase 1
Clinical Phase 2
Clinical Phase 3
Filing and registration
Post marketing activities
50-500 5-50 10-100 50-500
100-30000
100-1.109
0-80% response
0-80% response
0-70% response
Dose 1
Dose 2
Placebo
Comparator*
100 77 60 15 5 11000Compounds
Success rates
Current Pharmaceutical R&D
Few years
after launch
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Success rates per organ system
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https://www.nature.com/articles/nrd3405 From Pammolli et al. Nature Reviews Drug Discovery 2011
Target Discovery / Selection
Lead finding
Lead optimization
Pre-clinical development
Clinical Phase 1
Clinical Phase 2
Clinical Phase 3
Filing and registration
Post marketing activities
50-500 5-50 10-100 50-500
100-300000-10%SAE
0-15%SAE
1-20%SAE
Side effects
Current Pharmaceutical R&D
100-1.109
Few years
after launch
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Successful Drug discovery and development is team work!
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When and why did we start talking about biomarkers?
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Has biomarker research reached a plateau?
Biomarkers &trends
Reduced interest in 2018?
White paper from the FDA
Start
Human
Genome
project
Pe
rcen
tage B
iom
ark
er
cita
tio
ns in P
ub
med
year
24 April 2014COST CliniMARK Summerschool | 24 Sep 201919
From the 2018 FDA drug approvals you may think we’re uphill again
From Mullard, Nature Reviews Drug Discovery (2019)
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https://www.nature.com/articles/d41573-019-00014-x
…Moore‘s law in reverse: halving the output every 9 years
Eroom‘s law
From Scannell, Nature Reviews Drug Discovery (2012)
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https://www.nature.com/articles/nrd3681
Pharmaceutical R&D in the 21st Century: professional gambling or industrialized science?
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From Research inventions to Clinical Benefit
Valley of Death
From Butler, Nature 2008
Foundationn Meeting DayOne, Basel, 2016
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• Traditionally clinical diagnosis is based on symptomology
• Clinical endpoints are often not objective (think about pain)
• Outside of Oncology most diseases (except Mendelian disorders with a clear genetic
cause) do not use molecular information for diagnosis or classification
• Most of the current medications are not curative, they treat symptoms, not the cause
• Dosing of drugs are mostly based on an average response and an average safety level
• Adjusted dosing is mostly empirical
But…also medical practice can be inaccurate and imprecise
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What are the solutions to our problem?
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Wanted: new toolkit“A new product development toolkit - containing powerful
new scientific and technology methods such as animal or
computer-based predictive models, biomarkers for safety and
effectiveness and new clinical evaluation techniques - is
urgently needed to improve predictability and efficacy along
the critical path from lab concept to commercial product.”
FDA
FDA report, 2004
Janet Woodcock, CDER FDA
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https://www.fda.gov/science-research/critical-path-
initiative/critical-path-opportunities-reports
The answer to it was implementing the 5R’s
AstraZeneca’s painful soul searching of project failures
From Cook et al, Nature Reviews Drug Discovery 2014
COST CliniMARK Summerschool | 24 Sep 201928 http://www.nature.com/nrd/journal/v13/n6/pdf/nrd4309.pdf
• Exposure at the target site of action over a desired
period of time
• Binding to the pharmacological target as expected
for its mode of action
• Expression of pharmacological activity
commensurate with the demonstrated target
exposure and target binding
Pfizer’s three pillars of survival
Pfizer’s similar answer after a similar exercise
From Morgan et al., Drug Discovery Today 2012
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https://www.sciencedirect.com/science/article/pii/S1359644611004776
Interim Question:Do you know one of the bigger problems causing drugs not to work (or cause SAEs)?
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• Compliance has always been a big problem for oral administered drugs like antibiotics,
statins, anti inflammatory drugs, anti depressants, anti psychotics e.o.
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Compliance!!!
Systematic Biomarker Approaches
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Problem statement: what question are we trying to solve?
Biomarker Definition
• “A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”
Procedural Definitions
• Validation: technical, for the device and/or assay
• Qualification: the actual clinical utility
Biomarker types
• Susceptibility/risk
• Diagnostic
• Monitoring
• Prognostic
• Predictive
• Pharmacodynamic/response
• Safety.
Biomarker use
• Fit for Purpose
• Context of Use (COU)
Current definitions and semantic standards from traditional biomarkers
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https://www.ncbi.nlm.nih.gov/books/NBK326791/
Key decision points in pharmaceutical R&D
First in human
Including assays for key biomarkers (e.g. target engagement, pharmacodynamics)
Proof of Principle
Translational endpoints that provide evidence of relevant pharmacology activity (for internal decision making)
Proof of Concept
Clinical endpoint as close to registration endpoint as possible
Approval and
launch
• Tools to enable earliest possible assessment hypothesis-driven validated targets
• Drive attrition into Discovery and Early Clinical Development• Make data-driven decisions to assess relevant activity prior to
late stage development
Candidate
approval
Supporting biomarkers identified
Lead op
Identify translational
biomarkers
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Biomarker considerations: the reality of standard of care
EEG
Speech
UrineCSF
Whole blood
Imaging
ECG
Hair Follicles
Biopsies
Bone
Proteins/Metabolites
Skin
Any tissue•Non-invasive
•Moderate invasive
•invasive
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Biomarkers have different purposes in the R&D process and require different levels of
control
Fit-for-purpose principle
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Case Example 1
Pharmacodynamic Response Biomarker
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Biomarker support for Lead Optimization to Clinical Proof of Concept
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Amaravadi (2016) BioanalysisCOST CliniMARK Summerschool | 24 Sep 201940
Serotonin biosynthesis and homeostasis
AADC
TRP
5-HTP 5HT
Vascular tone
Gut motilityPrimary hemostasis
Cell mediated immune response
Migration, proliferation
Peripheral 5HT
MoodSleep
Anxiety
Food intake…..
Central 5HT
TPH
Rate limiting enzyme
Drug
other pathways inc. kynurenine
95%
5%
5-HT, serotoninTrp, tryptophanTPH, tryptophan hydroxylase
5-HT cannot cross the blood brain barrier and the CNS makes 5-HT locally using a CNS specific TPH isoformCOST CliniMARK Summerschool | 24 Sep 201941
• Carcinoid syndrome
− Carcinoid syndrome is caused by secretion of large quantities of 5-HT and other hormones from neuroendocrine tumors.
− Activation of 5-HT2b results in mesenteric and cardiac fibrosis leading to valvulopathy.
− The TPH1 inhibitor telotristat ethyl was FDA approved for treatment of carcinoid syndrome in 2017 (Marketed bx Lexicon Pharmaceuticals Ltd as Xermelo).
• Pre-clinical and translational studies indicate therapeutic potential of peripheral 5-HT synthesis inhibitors in diseases including
− Lung fibrosis
− Pulmonary arterial hypertension
− Ulcerative colitis
− Obesity
Peripheral Serotonin synthesis as a drug target
5-HT, serotoninTPH, tryptophan hydroxylase
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3 years of unsuccessful chemistry to find promising leads
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• Blood 5-HT rat t1/2 = 3 d, dog t1/2 = 6 d
Blood 5-HT has a long half-life
Blood Trp (M) Blood 5-HT (M)
- Trp - Trp
0 2 4 0 2 4
Need for a tracer
Measure 5-HT synthesis/ In vivo TPH1 activity
Trp
Trp 5-HT5-Hydroxytryptophan
5-HT, serotoninTrp, tryptophanTPH, tryptophan hydroxylase
COST CliniMARK Summerschool | 24 Sep 201944
• 13C, 15N labels, No deuteriums to avoid altered metabolism
• > 2 labels to avoid interference from naturally occurring +1 (10%) and +2 (1%) isotopes
(13C)
• Commercial availability/cost
Tracer selection is important
13C11,15N2-TRP was the only
One that fit all the criteria at the time
Cost per rat in an experiment ~20CHF
Estimated cost per human ~ 2000CHF
COST CliniMARK Summerschool | 24 Sep 201945
Tracer Unaltered
metabolism
Not
radioactive
Best data
quality
Fluoro-TRP
14C-Me-TRP
Heavy-TRPExact Mass = 204
Stable isotope tracer approach
Heavy-Trp = 13C11-Trp
MeTrp PET tracer is used clinically to measure 5-HT
synthesis, but results are confounded by Trp uptake
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Heavy serotonin measurement
h-TRP h-5-HTTPH1
TRP 5-HTTPH1
Exact Mass = 204 Exact Mass = 176
Exact Mass = 215 Exact Mass = 186
Welford, Vercauteren (2016) SciRep
5-HT, serotoninTrp, tryptophanTPH, tryptophan hydroxylase
t im e (h )
blo
od
Trp
(
M)
0 5 1 0 1 5 2 0 2 5
0
2 0
4 0
6 0
8 0
Trp
t im e (h )
blo
od
5-H
T (
M)
0 5 1 0 1 5 2 0 2 5
0
5
1 0
5 -H T
t im e (h )
blo
od
5-H
IAA
(n
M)
0 5 1 0 1 5 2 0 2 5
0
2 0
4 0
6 0
8 0
5 - H IA A
t im e (h )
blo
od
%(1
3C
11)T
rp
0 5 1 0 1 5 2 0 2 5
0
1 0
2 0
3 0
4 0
5 0
% (1 3
C 1 1 )T rp
t im e (h )
blo
od
%(1
3C
10)5
-HT
0 5 1 0 1 5 2 0 2 5
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
% (1 3
C 1 0 )5 -H T
t im e (h )
blo
od
(1
3C
10)5
-HIA
A (
nM
)
0 5 1 0 1 5 2 0 2 5
0
5
1 0
1 5
(1 3
C 1 0 )5 -H IA A
0 h O ra l
(13
C 11 )T r p
0 1 h 2 h
4 h6 h
2 4 h
B lo o d c o lle c t io n
D ru g a n d h -5 -H T
r e a d -o u t
COST CliniMARK Summerschool | 24 Sep 201948
h-5-HT synthesis in healthy rats
t im e (h )
blo
od
Trp
(
M)
0 5 1 0 1 5 2 0 2 5
0
2 0
4 0
6 0
8 0
Trp
t im e (h )
blo
od
5-H
T (
M)
0 5 1 0 1 5 2 0 2 5
0
5
1 0
5 -H T
t im e (h )
blo
od
5-H
IAA
(n
M)
0 5 1 0 1 5 2 0 2 5
0
2 0
4 0
6 0
8 0
5 - H IA A
t im e (h )
blo
od
%(1
3C
11)T
rp
0 5 1 0 1 5 2 0 2 5
0
1 0
2 0
3 0
4 0
5 0
% (1 3
C 1 1 )T rp
t im e (h )
blo
od
%(1
3C
10)5
-HT
0 5 1 0 1 5 2 0 2 5
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
% (1 3
C 1 0 )5 -H T
t im e (h )
blo
od
(1
3C
10)5
-HIA
A (
nM
)
0 5 1 0 1 5 2 0 2 5
0
5
1 0
1 5
(1 3
C 1 0 )5 -H IA A
0 h O ra l
(13
C 11 )T r p
0 1 h 2 h
4 h6 h
2 4 h
B lo o d c o lle c t io n
D ru g a n d h -5 -H T
r e a d -o u t
% l
ab
el
inc
orp
ora
tio
n
0
2
4
6
8 brain
% (13
C 11 )T rp
% (13
C 10 )5 -H T
% (13
C 10 )5 -H IAA
colon duodenum lung spleen thymus
0 h o ra l
(13
C 11 )T r p
0
1 0 h
s a c r if ic e a n d
o r g a n s a m p lin g
Blood
Tissue
Welford, Vercauteren (2016) SciRep
5-HT, serotoninTrp, tryptophan5-HIAA, 5-hydroxyindole acetic acid
n = 4AV±SD
n = 5AV±SD
COST CliniMARK Summerschool | 24 Sep 201949
TPH inhibitor pharmacodynamics with oral h-Trp
t im e (h )
blo
od
%(1
3C
11)T
rp
0 5 1 0 1 5 2 0 2 5
0
1 0
2 0
3 0
4 0
5 0 V ehic le
30m g/kg PC PA
100m g/kg PC PA
% (13
C 11 )T rp
t im e (h )
blo
od
%(1
3C
10)5
-HT
0 5 1 0 1 5 2 0 2 5
0 .0
0 .5
1 .0
1 .5
2 .0
% (13
C 10 )5 -H T
t im e (h )
blo
od
5-H
IAA
(n
M)
0 5 1 0 1 5 2 0 2 5
0
2 0
4 0
6 0
8 0
1 0 0
5-HIAA
tim e (h )
blo
od
Trp
(
M)
0 5 1 0 1 5 2 0 2 5
0
2 0
4 0
6 0
8 0
1 0 0
T rp
t im e (h )
blo
od
5-H
T (
M)
0 5 1 0 1 5 2 0 2 5
0
5
1 0
1 5
2 0
5 -H T
t im e (h )
blo
od
(1
3C
10)5
-HIA
A (
nM
)
0 5 1 0 1 5 2 0 2 5
0
2
4
6
8
1 0
1 2
(13
C 10 )5-HIAA
D ru g
-1 h
0 h O ra l
(13
C 11 )T r p
0 1 h 2 h
4 h6 h
2 4 h
B lo o d c o lle c t io n
D ru g a n d h -5 -H T
r e a d -o u t
Welford, Vercauteren (2016) SciRep
-PCPA is a non-specific brain penetrating TPH inhibitor
-h-5-HT is the most sensitive marker of TPH inhibition in rats5-HT, serotoninTrp, tryptophan
5-HIAA, 5-hydroxyindole acetic acidPCPA, para-cholorophenylalanine
n = 4AV±SD
COST CliniMARK Summerschool | 24 Sep 201950
TPH inhibitor pharmacodynamics with infused h-Trp
t im e (h )
blo
od
%(1
3C
11)T
rp
0 2 4 6 8 1 0
0
2
4
6
8
% (13
C 11 )T rp
V ehic le
6m g/kg
20m g/kg
60m g/kg
te lo tr istat
ethyl
t im e (h )
blo
od
%(1
3C
10)5
-HT
0 2 4 6 8 1 0
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
% (13
C 10 )5 -H T
t im e (h )
blo
od
(1
3C
10)5
-HIA
A (
nM
)
0 2 4 6 8 1 0
0
5
1 0
1 5
2 0
(13
C 10 )5-HIAA
t im e (h )
blo
od
Trp
(
M)
0 2 4 6 8 1 0
0
2 0
4 0
6 0
8 0
T rp
t im e (h )
blo
od
5-H
T (
M)
0 2 4 6 8 1 0
0
2
4
6
8
5 -H T
t im e (h )
blo
od
5-H
IAA
(n
M)
0 2 4 6 8 1 0
0
2 0
4 0
6 0
5-HIAA
D ru g
-0 .5 h
0 h b o lu s
(13
C 11 )T r p
0 1 h 2 h
4 h6 h
8 h
B lo o d c o lle c t io n
D ru g a n d h -5 -H T
r e a d -o u t
C o n s ta n t (13
C 11 )T r p in fu s io n
Welford, Vercauteren (2016) SciRep
5-HT, serotoninTrp, tryptophan5-HIAA, 5-hydroxyindole acetic acid
n = 6AV±SD
-Telotristat ethyl dose dependently reduces h-5-HT
production in rats
-h-Trp infusion allows estimation of duration of action
COST CliniMARK Summerschool | 24 Sep 201951
Translating the TPH1 inhibition effects
? ?
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• 6 healthy subjects per group: 3 males and 3 females
• Subjects were overnight fasted up to 4 h post dose (lunch)
• The 70 and 250 mg cohorts were administered on the same day, the 700 mg cohort
several months later
Heavy tryptophan study in man: Design
Screening-21/-2 days-21/-11 days
PK samples over 24 h
Day 1:h-Trp
Day 31-33:SAE follow-up
Administered doses:
70, 250, 700 mg
Gehin, Welford (2016) submittedCOST CliniMARK Summerschool | 24 Sep 201954
h-5-HT synthesis in healthy subjects
Gehin, Welford (2016) submitted
5-HT, serotoninTrp, tryptophan5-HIAA, 5-hydroxyindole acetic acid
COST CliniMARK Summerschool | 24 Sep 201955
D o g
1 2 m g /k g h -T R P
t im e (h r )
blo
od
TR
P(u
M)
blo
od
h-5
-HT
an
dh
-5-H
IAA
(nM
)
0 5 1 0
0
2 0
4 0
6 0
0
1 0
2 0
3 0
4 0
R a t
3 0 m g /k g h -T R P
t im e (h r )
blo
od
TR
P(u
M)
blo
od
h-5
-HT
an
dh
-5-H
IAA
(nM
)
0 5 1 0
0
2 0
4 0
6 0
0
5 0
1 0 0
1 5 0
2 0 0
M a n
3 .2 m g /k g h -T R P
t im e (h r )
blo
od
TR
P(u
M)
blo
od
h-5
-HT
an
dh
-5-H
IAA
(nM
)
0 5 1 0
0
2 0
4 0
6 0
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
D o g
1 2 m g /k g h -T R P
t im e (h r )
blo
od
TR
P(u
M)
blo
od
h-5
-HT
an
dh
-5-H
IAA
(nM
)
0 5 1 0
0
2 0
4 0
6 0
0
1 0
2 0
3 0
4 0
R a t
3 0 m g /k g h -T R P
t im e (h r )
blo
od
TR
P(u
M)
blo
od
h-5
-HT
an
dh
-5-H
IAA
(nM
)
0 5 1 0
0
2 0
4 0
6 0
0
5 0
1 0 0
1 5 0
2 0 0
M a n
3 .2 m g /k g h -T R P
t im e (h r )
blo
od
TR
P(u
M)
blo
od
h-5
-HT
an
dh
-5-H
IAA
(nM
)
0 5 1 0
0
2 0
4 0
6 0
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
• h-5-HT changes more dramatically than 5-HT between species
• In man synthesis is linear despite rapidly changing h-TRP, i.e. h-5-HT production in a
single compartment will not explain the data
h-5-HT production changes dramatically with species
0.3 kg 15 kg 80 kgWeight
73 M 54 M 48 MTrp
13 M 5.0 M 1.7 M5-HT
Blo
od
h-T
RP
(
M)
Blo
od
h-T
RP
(
M)
Blo
od
h-T
RP
(
M)
~
Gehin, Welford (2016) submitted
5-HT, serotoninTrp, tryptophan5-HIAA, 5-hydroxyindole acetic acidCOST CliniMARK Summerschool | 24 Sep 201957
Acknowledgements
R Welford
A Trébaul
M Garzotti
D Eckert
F Griaud
S Glutz
L Remen
M Vercauteren
C Cattaneo
G von Aesch
E Fournier
O Nayler
P Sieber
S Roux
M Probst
J Segrestaa
C Kohl
M Machacek (Lyo-X)
M Gehin-Beurne
& team
J Dingemanse
R Karge
COST CliniMARK Summerschool | 24 Sep 201960
COST CliniMARK Summerschool | 24 Sep 201961
http://dx.doi.org/10.1038/srep30059 http://doi.wiley.com/10.1002/cpt.1087
Case example 2
Diagnostic Biomarker
COST CliniMARK Summerschool | 24 Sep 201962
Plasma lyso-sphingomyelin and glucosylsphingosine as biomarkers for Niemann-Pick disease type C
COST CliniMARK Summerschool | 24 Sep 201963
Crocker 1961 J. Neuro Chem
NP-C is a sphingolipidosis
“The clinical picture in Niemann-Pick disease, as the syndrome is conventionally
defined, is variable. The table summarizes the cumulative experience in this hospital
with patients who show a constitutional affliction by major visceral sphingomyelin
accumulation in the usual type of “foam cell”.”
COST CliniMARK Summerschool | 24 Sep 201964
Degradation of sphingolipids
NP-C: Accumulation of
Sphingomyelin, Glucosylceramide,
Lactosylceramide, GM2 and GM3
Vanier (2010) Orphanet
Figure adapted from
©www.lysosomalstorageresearch.org
COST CliniMARK Summerschool | 24 Sep 201965
Plasma lyso-sphingolipids in LSDs
DiseaseSphingolipid(s)
accumulated in organsLyso-sphingolipid in plasma
References for lyso-form as a biomarker
Fabry Gb3lysoGb3 >10-fold Aerts et al. (2008) ,Krüger et
al (2012), Togawa et al.
(2011)
Krabbe Galactosylceramide Psychosine 10-foldZhu et al. (2012),
Svennerholm et al. (1980)
Gaucher Glucosylceramide Glucosylsphingosine 100-fold Dekker et al. (2011),
Tay-Sachs/Sandhoff GM2 lysoGM2 10-fold Kodama et al. (2011)
NP-C
Sphingomyelin
Glucosylceramide
Lactosylceramide
GM2 - GM3
Lyso-sphingomyelin (SPC) x-fold?
Glucosylsphingosine (GlcSph) x-fold?
Lactosylsphingosine x-fold?
lysoGM2 - lysoGM3 x-fold?
Sphingolipid Analogous lyso-sphingolipid
Vanier (2010). Orphanet J. Rare Dis.
Sandhoff (2013). Biochem. Soc. Trans.
COST CliniMARK Summerschool | 24 Sep 201966
Feasibility study : ZOOM study
ZOOM was a multi-center genetic screening study on the occurrence of NP-C in adult patients with
neurological/psychiatric symptoms.
Of the screened patients, 3 of 250 were identified as NP-C positive based on genetics and filipin staining
Bauer et al. Hum. Mol. Genet. (2013)
S P C
pla
sm
a c
on
ce
ntr
ati
on
(n
M)
NP
-C n
eg
at i
ve
NP
-C u
ncer t
ain
NP
-C p
osit
ive
0
2 0
4 0
6 0
8 0
1 0 0
G lu c o s y ls p h in g o s in e
pla
sm
a c
on
ce
ntr
ati
on
(n
M)
NP
-C n
eg
at i
ve
NP
-C u
ncer t
ain
NP
-C p
osit
ive
0
5
1 0
1 5
COST CliniMARK Summerschool | 24 Sep 201967
Technical LC-MS/MS Assay validation
24 April 2014COST CliniMARK Summerschool | 24 Sep 201968
• EMA guideline on analytical method validation
(EMEA/CHMP/EWP/192217/2009) (2011)
• FDA: Guidance for Industry Bioanalytical Method Validation (2001)
• Jennings et al. “Recommended principles for validating clinical molecular
pathology tests” (2009) Arch. Path Lab. Med.
• Houghton, R., et al., Generic approach to validation of small-molecule LC-
MS/MS biomarker assays. Bioanalysis, 2009. 1(8): p. 1365-74.
Source documents
technical assay validation
COST CliniMARK Summerschool | 24 Sep 201969
Validation plan: CAL and qc samples
AnalyteQC2
plasma
QC3
plasma spiked
QC4
plasma spiked
[nM] [nM] [nM]
SPC X1 100 + X1 360 + X1
GlcSph X2 10 + X2 36 + X2
Analyte CAL1 CAL2 CAL3 CAL4 CAL5 CAL6 CAL7 CAL8 CAL9
[nM] [nM] [nM] [nM] [nM] [nM] [nM] [nM] [nM]
SPC 5 10 25 50 100 160 250 360 500
GlcSph 0.5 1 2.5 5 10 16 25 36 50
aliquots
in barcoded storage tubes
frozen at –20°C.
Houghton et al Bioanalysis.2009
CAL samples are prepared by spiking standard SPC and GlcSph in 10 fold diluted plasma (PBS)
COST CliniMARK Summerschool | 24 Sep 201970
precision: 3 batches
SPCQC2 QC3 QC4
CV [%] 8.6 3.2 4.2
GlcSph QC2 QC3 QC4
CV [%] 15.0 4.8 5.7
COST CliniMARK Summerschool | 24 Sep 201971
Accuracy: 3 batches
SPCQC2 QC3 QC4
accuracy [%] 100.0 85.0 95.7
GlcSphQC2 QC3 QC4
accuracy [%] 100.0 108.8 115.6
COST CliniMARK Summerschool | 24 Sep 201972
Robustness: Validation on different LCMS platforms
DIONEX
QTRAP
6500
DIONEX
QTRAP
5500
SHIMAZU
QTRAP
API4000
COST CliniMARK Summerschool | 24 Sep 201973
Robustness: QCs on different LCMS platforms
API
4000
QTRAP
5500
QTRAP
6500
API
4000
QTRAP
5500
QTRAP
6500
+15%
-15%
+15%
-15%
+20%
-20%
+15%
-15%
+15%-15%
+20%
-20%
COST CliniMARK Summerschool | 24 Sep 201974
Short term Stability of Qcs
S P C : s ta b ility o f Q C s
% a
cc
ura
cy
roo
m t
em
p (
4h
rs)
roo
m t
em
p (
20h
rs)
3 f
reeze-t
haw
s c
ycle
s
24h
r au
tosam
ple
r
8 0
9 0
1 0 0
1 1 0
1 2 0
Q C 2
Q C 3
Q C 4
G lc S p h : s ta b ility o f Q C s
% a
cc
ura
cy
roo
m t
em
p (
4h
rs)
roo
m t
em
p (
20h
rs)
3 f
reeze-t
haw
s c
ycle
s
24h
r au
tosam
ple
r
8 0
9 0
1 0 0
1 1 0
1 2 0
Q C 2
Q C 3
Q C 4
mean±SD (n = 3)
COST CliniMARK Summerschool | 24 Sep 201975
Long term Stability of Qcs
-Issue observed with QC2 for SPC at >50days when stored at -20°C
-But, 2yr old samples stored at -80°C are in the normal range
d a y s
co
nc
en
tra
tio
n (
nM
)
0 2 5 635
36
40
48
79
91
92
93
138
142
4
6
8
1 0
1 2
1 4
d a y s
co
nc
en
tra
tio
n (
nM
)
0 2 5 635
36
40
48
79
91
92
93
138
142
6 0
8 0
1 0 0
1 2 0
d a y s
co
nc
en
tra
tio
n (
nM
)
0 2 5 635
36
40
48
79
91
92
93
138
142
3 0 0
4 0 0
5 0 0
6 0 0
d a y s
co
nc
en
tra
tio
n (
nM
)
0 2 5 635
36
40
48
79
91
92
93
138
142
0 .4
0 .6
0 .8
1 .0
d a y s
co
nc
en
tra
tio
n (
nM
)
0 2 5 635
36
40
48
79
91
92
93
138
142
8
1 0
1 2
1 4
1 6
d a y s
co
nc
en
tra
tio
n (
nM
)
0 2 5 635
36
40
48
79
91
92
93
138
142
3 0
3 5
4 0
4 5
5 0
5 5
SPC
GlcSph
QC2 valav±20% QC3 valav±15% QC4 valav±15%
mean±SD (n = 2-6)COST CliniMARK Summerschool | 24 Sep 201976
Plasma stability: 5 donors, 96hrs
SPC
GlcSph
4°C Room temp
t im e (h rs )
pla
sm
a S
PC
(n
M)
0 2 0 4 0 6 0 8 0
5
6
7
8
9
1 0
t im e (h rs )
pla
sm
a S
PC
(n
M)
0 2 0 4 0 6 0 8 0
5
6
7
8
9
1 0
t im e (h rs )
pla
sm
a G
lcS
ph
(n
M)
0 2 0 4 0 6 0 8 0
0
1
2
3
t im e (h rs )
pla
sm
a G
lcS
ph
(n
M)
0 2 0 4 0 6 0 8 0
0
1
2
3
4
d o n o r 1 0
d o n o r 1 1
d o n o r 1 2
d o n o r 1 3
d o n o r 1 4
tlast-tfirst = +0.3%
tlast-tfirst = +0.8%
tlast-tfirst = +13%
tlast-tfirst = +17%
COST CliniMARK Summerschool | 24 Sep 201977
Whole Blood stability: 3 donors, 5 hours, room temperature
tlast-tfirst = +4.5%
S P C
t im e (m in s )
pla
sm
a S
PC
(n
M)
0 1 0 0 2 0 0 3 0 0
5 .5
6 .0
6 .5
7 .0
7 .5
8 .0
G lc S p h
t im e (m in s )
pla
sm
a G
lcS
ph
(n
M)
0 1 0 0 2 0 0 3 0 0
0 .0
0 .5
1 .0
1 .5
2 .0
d o n o r 1
d o n o r 2
d o n o r 3
tlast-tfirst = +3.8%
COST CliniMARK Summerschool | 24 Sep 201978
Application to NP-C patient samples
COST CliniMARK Summerschool | 24 Sep 201979
• 1 sample per subject
• NP-C samples
− From 3 centers
• Control samples
− From 2 centers and one
commercial provider
Retrospective case control analysisDD mmm YYYY
Group Control NP-C
Number of values 55 57
Male (%) 42 44
Female (%) 58 56
Age (yrs) 25% percentile 9 10.5
Median age (yrs) 14 15
Age (yrs) 75% percentile 23 22
Receiving miglustat (%) 44
Miglustat naive (%) 40
Unknown miglustat status (%) 16
COST CliniMARK Summerschool | 24 Sep 201980
SPC and GlcSPHDD mmm YYYY
P la s m a S P C (n M )p
las
ma
SP
C (
nM
)
co
nt
(n =
55)
NP
-C (
n =
57)
0
2 0
4 0
6 0
P la s m a G lc S p h (n M )
pla
sm
a G
lcS
ph
(n
M)
co
nt
(n =
55)
NP
-C (
n =
57)
0
5
1 0
COST CliniMARK Summerschool | 24 Sep 201981
DD mmm YYYY
0-50 years, miglustat naïve
patients, n =23
Controls, n = 55
Area = 0.992
Cut-point of 10.6nM separates
control from NP-C with a sensitivity
of 96% and a specificity of 95%
R O C o f p la s m a S P C
m ig lu s ta t n a iv e N P C p a tie n ts o n ly
F a ls e p o s it iv e ra te (1 - S p e c if ic ity )
Tru
e p
os
itiv
e r
ate
(s
en
sit
ivit
y)
0 .0 0 .2 0 .4 0 .6 0 .8 1 .0
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
Area under the ROC curve
Area
Std. Error
95% conf idence interval
P value
Results
0.9921
0.007741
0.9769 to 1.007
< 0.0001
> 11.54
Sensitivity
0.9565
95% CI
0.7805 to 0.9989
Specif icity
0.9818
95% CI
0.9028 to 0.9995
Likelihood ratio
52.61
COST CliniMARK Summerschool | 24 Sep 201982
PPV and NPV• Positive predictive value (PPV)- “What is the likely hood that the patient has the disease when the
test result is positive”
• Negative predictive value (NPV)- “The probability that the patient does not have the disease when the result is negative”
• Context of use and the decisions to be made are important for understanding usefulness of a test (note sens and spec also need to be determined in right setting)
• In the case of NP-C combination of neurological symptoms, BMs and genetics
• In broad screening there will be many false positives even with a high specificity diagnostic assay
prevalence sensitivity specificity PPV NPV
0.05 0.9565 0.9565 0.5365 0.9976
0.01 0.9565 0.9565 0.1817 0.9995
0.00001 0.9565 0.9565 0.0002 1.0000
0.05 1 0.97 0.6369 1.0000
0.01 1 0.97 0.2519 1.0000
0.00001 1 0.97 0.0003 1.0000
COST CliniMARK Summerschool | 24 Sep 2019
acknowledgements
Idorsia
Richard Welford
Marco Garzotti
Andreas Brecht
Joerg Velker
Stefan Kolb
Olivier Morand
Jasper Dingemanse
Martine Gehin-Beurne
Patricia Sidharta
Andreas Krause
For the SPC clinical qualification:
Hospital das Clínicas de Ribeirão PretoSão Paulo, Brazil
Dr Charles Marques Lourenço
Villa Metabolica
Mainz, Germany
Dr Eugen MengelUniversitätsklinikum
Münster, Germany
Dr Thorsten MarquardtDr Janine Reunert
COST CliniMARK Summerschool | 24 Sep 201985
COST CliniMARK Summerschool | 24 Sep 201986
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114669
Avoid the stereo typing, pharma is working intensively with the community
Collaboration with Rare Disease Centers
COST CliniMARK Summerschool | 24 Sep 201987
Case example 3
Pharmacodynamic Response Biomarker: Target Engagement
COST CliniMARK Summerschool | 24 Sep 201988
• Paracelsus: "All things are poison, and nothing is without poison, the dosage alone makes it so a thing is not a poison.“
• Searching for new drugs requires establishing a clear safety window. Thispreferably is muchbigger than the therapeutic window
Therapeutic vs. Safety window
COST CliniMARK Summerschool | 24 Sep 201989
For drugs it’s all about the dose and the effect
Ex
po
sure
(d
rug
co
nce
ntr
ati
on
in b
loo
d o
r ti
ssu
e)
Sa
fety
win
do
w
Ph
arm
aco
dyn
am
ic e
ffe
ct
Pharmacodynamic and Target engagement in the search for a potent CRTH2 antagonist for seasonal allergic rhinitis
COST CliniMARK Summerschool | 24 Sep 201990
• GPCR signalling is a complex process. One of the ways some GPCR signalling is
controlled in a feedback loop is through internalization.
Regulation of signalling of GPCRs is complex
Developing a flow cytometer assay for receptor internalization
From: Ramachandran et al. Nature Reviews Drug Discovery 2012
Receptor internalization is a very proximate target engagement biomarker and in principle
also a pharmacodynamic, downstream signalling marker
Measuring receptor levels ex vivo reveals a dynamic view on relation between exposure
(PK) and effect (PD)
COST CliniMARK Summerschool | 24 Sep 201991
Receptor Occupancy Assay Principle
receptorantagonist agonist
Whole blood
leukocytes
staining antibody
Stimulation StainingFlow cytometric
measurement
COST CliniMARK Summerschool | 24 Sep 201992
Normalization
normalization
Pharmacodynamic (PD) effect
COST CliniMARK Summerschool | 24 Sep 201993
Biomarker Selection - Precision
DD mmm YYYY
CRTH2
internalization
CD11b
upregulationCRTH2 internalization
CD11b upregulation
COST CliniMARK Summerschool | 24 Sep 201994
Biomarker Selection - Feasibility
DD mmm YYYY
GPCR internalization assay on PBMCs.
applying intrinsic normalization to calculate the PD effect.
Actelion Blood Donation Day:
- feasibility
- throughput
- normalization
max min PD
COST CliniMARK Summerschool | 24 Sep 201996
COST CliniMARK Summerschool | 24 Sep 201997
Fit-for-Purpose Assay Validation
Outcome: reliable PD biomarker for GPCR antagonist efficiency
GPCR
Phase I PD Biomarker Results
SAD
MAD
800 mg o.d.
100 mg o.d.
10 mg o.d.
Placebo o.d.
February 24, 2015COST CliniMARK Summerschool | 24 Sep 201998
PK/PD modelling
Failed trial in Seasonal Allergic Rhinitis (SAR)
PK/PD model
determined dosing regimen
GO!
COST CliniMARK Summerschool | 24 Sep 201999
acknowledgements
Idorsia
Daniel Strasser
Herve Farine
Virginie Sippel
Martin Holdener
Martine Gehin-Beurne
Patricia Sidharta
Jasper Dingemanse
Andreas Krause
Jochen Zisowsky
COST CliniMARK Summerschool | 24 Sep 2019101
COST CliniMARK Summerschool | 24 Sep 2019102
http://doi.org/10.1007/s40262-015-0354-3
Another GPCR storyPharmacodynamic Biomarkers and Target Engagement
COST CliniMARK Summerschool | 24 Sep 2019103
SAD dose responsiveness of FPR2 internalization and TNFa
Results: SAD
COST CliniMARK Summerschool | 24 Sep 2019104
Daily
40, 200 800)
Dosing schedules and Doses TestedStudy drug administration
Qd2
(40mg)
Qd3
(200mg)
Day: D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13
Biomarker Sampling
02
1224
26
1224
0 0 0 2
1224
0 0 2
1224
0 0 2
1224
0 0 0
White
blood cell
count
0126
24
0126 Sputum
EOT
LPS
COST CliniMARK Summerschool | 24 Sep 2019105
target engagement marker
COST CliniMARK Summerschool | 24 Sep 2019106
Hematological effect on blood cell counts
results
Monocytesneutrophils
DA
Y 1
DA
Y 7
COST CliniMARK Summerschool | 24 Sep 2019107
Downstream marker: cytokines
STOP!
COST CliniMARK Summerschool | 24 Sep 2019108
COST CliniMARK Summerschool | 24 Sep 2019109
https://www.ncbi.nlm.nih.gov/pubmed/27730665
Biomarkers in the lab versus biomarkers on site
The case for assay transfer and monitoring
COST CliniMARK Summerschool | 24 Sep 2019110
Method familiarization
Training session in the lab• 2 fresh EDTA blood samples
• 2 to 3 different drug levels
• 1 vehicle
Presentation• assay protocol
• results obtained
• system suitability criteria
• highlighting of the critical
steps
1 2
• 5 technicians for the clinical site• 2 technicians for the flow cytometry facilities• 2 lab managers
Team
COST CliniMARK Summerschool | 24 Sep 2019111
Is it not going to cost even more?
COST CliniMARK Summerschool | 24 Sep 2019112
Costs is not the right question
One striking example which has been a dramatic difference between to similar concepts
Anti PD1 (immune checkpoint inhibitor)
BMS: OpdivoMerck: Keytruda
Having a biomarker early in the process to differentiate patients on PD1 levels turned to be a costly, yet profitable. Failure costed BMS significantly.
COST CliniMARK Summerschool | 24 Sep 2019113
Probability of clinical success significantly improves with biomarkers
COST CliniMARK Summerschool | 24 Sep 2019114
https://academic.oup.com/biostatistics/article/20/2/273/4817524 From Wong et al. Biostatistics 2018
Pharma is still the one of the biggest R&D investors
In 2017:Top 20 Pharma spent $93.7 Billion on R&D while public spending only reached: $51.2 Billion
COST CliniMARK Summerschool | 24 Sep 2019115
We have been improving for more than a decade, are we doing better?
COST CliniMARK Summerschool | 24 Sep 2019116
AstraZeneca, after implementing the 5Rs
From Morgan et al, Nature Reviews Drug Discovery 2018
COST CliniMARK Summerschool | 24 Sep 2019117 https://www.nature.com/articles/nrd.2017.244
Biomarkers increase the %POS
Source : Health Decisions CRO+ reports 2015
COST CliniMARK Summerschool | 24 Sep 2019118
• Limited biomarker clinical uptake is symptomatic for our current modern biomedical
research
• Translation of biomarker research into clinical benefits needs radical improvement on
several levels
• Specifically the pharma industry is also in great need for novel biomarkers in the path
from discovery to clinical benefit
• Robust scientific methodology does not solve all problems, but is key to success and to
avoid waste
• Remember: an invention does not qual innovation!
• Biomedical innovation is team work along the healthcare chain and between all parties,
profit and non-profit: let’s avoid polarizing discussions.
Summary
COST CliniMARK Summerschool | 24 Sep 2019119
Please read this book!
Cultural Changes Needed….
COST CliniMARK Summerschool | 24 Sep 2019120
Outlook for the Future
COST CliniMARK Summerschool | 24 Sep 2019121
The digital revolution in health care
COST CliniMARK Summerschool | 24 Sep 2019122
• Imagine when compliance and dosing could be followed real time!
First Digital pill approved in 2017 by the FDA
COST CliniMARK Summerschool | 24 Sep 2019123