Role of DMPK Platform
T h l
Technology
& Knowledge
K
l d
in Helping Early
Discovery Research
Andy Harrell
Property of GlaxoSmithKline
Discovery
y
Preclinical
Clinical
Non Clinical Development
Clinical
Discovery
y
Preclinical
Clinical
Regardless
R
dl
off organisation,
i ti
some aspects
t off DMPK are best
b t
deeply embedded within the discovery program, whilst others
are best operating as platforms across multiples programs.
A collection of Short Stories which
demonstrate how platform science and
knowledge can be used to help drug discovery
5
Definition of a Platform Science and Knowledge
An expensive piece of equipment or suite
of equipment...
...or ...
...specialist scientific knowledge/expertise
associated with only a few highly trained
individuals...
... possibly applied across the whole
organisation on most projects at some
stage...
. ...but not necessarily routinely applied
within a single project...
...often
often contributing through bespoke
support to answer a specific question.
Presentation title in footer
00 Month 0000
6
Drive Through Metabolism
by Steve Thomas & Nigel Deeks
Platform: Nuclear Magnetic Resonance Spectroscopy
Specialist Knowledge: Metabolite Identification
Drive Through
g Metabolism
Promising chemical scaffold.
(1) Bulk Microsome
Incubation
(2) Fractionation
Response
Clearance issue which could not be
resolved by mass spectrometry alone
Time
NMR
(3) Identification
+16
%
(4) Quantification
DRUG
70
METABOLITE
30
Drive Through
g Metabolism
Major routes of metabolism identified in very short
time-frame commensurate with drug
g discovery.
y
OH
Metabolite amounts estimated by NMR integral of a
common proton.
Methods also applicable to plasma, bile and urine
from animals and man.
Project team able to address pharmacokinetics
through structural modification.
OH
Evaluation of preparative high performance liquid chromatography and
cryoprobe-nuclear magnetic resonance spectroscopy for the early
quantitative estimation of drug metabolites in human plasma
G J Dear,
G.J.
D
AD R
A.D.
Roberts,C.
b t C B
Beaumont,S.E.
tSE N
North
th
Seeing is Believing
By Steve
B
S
Castellino,
C
lli
Bianca
Bi
Squillaci,
S ill i
William Hardesty, Reid Groseclose , Peter
Marshall and Josie Morrell
Platform: MALDI MS Imaging
Specialist Knowledge: Drug Disposition
Seeing is Believing
Co-crystallisation of
analyte(s) with matrix
Extraction of
analyte
into matrix
droplet
Matrix Application
Laser
Ion distribution map can be generated
for any ion of interest
MS Spectra
acquired across
tissue surface
Matrix applied evenly
across surface using
automated device
~ 12µm sections thaw-mounted
onto surface e.g.
e g glass slide
Frozen tissue
Sliced using
a cryo-microtome
Adjacent slide retained
for staining
H&E stained tissue
MS and H&E data overlaid in software
Seeing is Believing
MALDI-MS imaging has been used to help
the GSK effort to discover new drugs
g for
skin indications by...
...comparing the skin penetration of
various drugs & formulations..
... and visualising target engagement for a
sweat gland indication.
Seeing is Believing
MALDI-MS imaging has also been used to
help in the discovery of new drugs for
respiratory
i t
iindications.
di ti
e.g.
e
g by correlating drug
disposition with histology or
pharmacology
...or investigating
i
ti ti
accumulation in the animal
lungs
Day 1
Day 28
Too Hot to Handle
by Alison Churchill Ian Baines & Rob
Chambers
Platform: Radioisotopes
Specialist Knowledge: Mechanisms of Metabolism
Short Story
y 2: Too Hot to Handle
Targeted covalent binding via Michael
addition of αβ unsaturated carbonyl group.
group
Unprecedented mechanism within GSK
Program collecting as much information as
possible on specificity of binding.
[14C]
Early ADME using
material conducted
within DMPK prior to candidate selection
Covalent interaction with target.
Concerns about non-specific interactions
resulting in direct, genetic or immunogenic
toxicity.
SH
Too Hot to Handle
% Radioactive Dose (0-24 hours)
Urine
25
Faeces
57
Wash
2
Total
85
14C
Too Hot to Handle
Metabolism
Alerts
Trapping
In Vivo
Covalent Binding
CYP
In Vitro
In Vivo
QWBA
Body Burden
Tissue
Overall
Dose
Risk deemed moderate based on integrated weight of evidence assessment.
Need for low dose emphasised
p
to p
project
j
team.
First Time in Human included a dose level stopping criteria regardless of
systemic exposure.
Predictable Pathways
by Gary Collins.
Platform: Metabolic Prediction Software
Specialist Knowledge: Drug Metabolism; Computational Chemistry
Predictable Pathways
y
A suite of complementary software packages
which in the hands of an experienced user
which,
user, can
predict metabolic routes and advise on
structural modifications which may modify
safety, efficacy and pharmacokinetics.
MetaSite
M t Sit
CYP-mediated prediction
Predictable Pathways
y
Metabolite Browser and Meteor were used to
demonstrate risks associated with benzodiaxoles
(autoinhibition and reactive quinones as a result of
ring opening)
...as a result direction of
chemistry was diverted toward
active ethoxy structures...
...Metasite predicted O-dealkylation as most likely
route of metabolism. DEREK raised a genotoxicity
alert for corresponding quinone-imine.
Risk mitigated because no extractable proton on
tertiary nitrogen. Yet to establish whether Odealkylation is problematic from a PK view
view.
Predictable Pathways
y
Predicting Increasing Metabolic Stability
In this example MetaSite was used to help a discovery
programme optimise on metabolic stability
Additional advice provided on
aldehyde oxidase (see later on).
Gone with the Wind
by Helen Tracey, Stephanie North.
Platform: DMPK WIKI, In vitro assays.
Specialist Knowledge: Corporate History
History, Drug Metabolism
Metabolism.
Gone with the wind
N
N
N
H
Molecules looked progressable based on rat, dog in
vivo clearance & microsomal clearance (all species).
Moderate - high clearance in cynomolgus monkey.
>
>
Gone with the wind
N
N
Aldehyde Oxidase (AO)
N
H
Previous experience with AO recalled.
non microsomal non
CYP enzyme prevalent in man
AO is a non-microsomal,
non-CYP
man.
Substrates usually have good bioavailability in rat & dog and are
stable in microsomes.
AO substrates, however, often have poor PK in man.
AO screen applied to help progress molecules without AO liability
liability.
Gone with the wind
A considerable amount of information was available on DMPK
WIKI (a DMPK knowledge depository similar to wikipedia) which
covered both past experiences and scientific information.
Examples of regio-selective
oxidation by aldehyde oxidase.
Electron deficiencies correlate with regioselective metabolism - numbers in red highlight
position(s)
p
( ) of oxidation.
Challenging Journeys
by Nainesh
b
i
h Patel,
l Donna Fraser, David
id
Kenworthy, Richard Snell
Platforms: Chromatography and Mass Spectrometry
Specialist Knowledge: Oligonucleotide and peptide analysis in biological
matrices
Challenging
g g Journeys
y
Delivery to target tissue remains a key problem for oligonucleotide discovery
programs across a variety of disease indications
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
U
A
G
C
DMPK was asked to help investigate metabolic stability and distribution of
base, linker and conjugate modifications for an anti-sense therapy.
Challenging
g g Journeys
y
DPM
180.00
160.00
140.00
120.00
100.00
80.00
60 00
60.00
40.00
Target tissue extract
20.00
0.00
0.00
10.00
20.00
30.00
40.00 mins
Target tissue extract
Oligonucleotide
& Metabolites
Target Concentration vs. Time
Using radiolabel and cold approaches
concentrations were measured &
compared concentrations for each
separate analogue in target tissues in
order to assess the effectiveness of
the structural modification.
Tissue Concen
ntration / [ng/g
g]
6E+4
5E+4
4E+4
3E+4
2E+4
2E 4
1E+4
0E+0
0
50
Group 1
100
Time / Hours
Group 2
150
Group 3
This example draws upon people with
deep seated analytical expertise able
deep-seated
to rise to the challenge of many
different types of molecules.
Challenging
g g Journeys
y
For a different discovery programme looking at short
i t f i RNA ((siRNA),
interfering
iRNA) th
the activity
ti it seen iin cellll lilines
could not be replicated in vivo.
We were able to compare
p
metabolic vulnerability
y of
different anti-sense strands in the relevant biofluid.
Doubtful (also) whether the siRNA was ever present,
i vivo,
in
i
in
i the
th duplex
d l fform required
i d ffor activity.
ti it
70% Turnover of various siRNA oligonucleotides
60
50
40
Sense
Anti-sense
30
20
10
0
OG1
OG2
OG3
OG4
OG5
OG6
Challenging Journeys
Serum stability was an import consideration for a discovery
unit trying to engineer proteolytically resistant peptides that
can enter cells and modulate protein interactions
Drawing on DMPK experience of peptide analysis, DMPK
developed MS assays which compared serum stability for 33
different peptide analogues
analogues.
Some peptide sequences and conformations were clearly
more stable than others providing a clear structural steer for
the discovery unit to follow
follow.
0.60
0.50
0.40
0.30
0.20
0.10
0.00
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500
1000
1500
Time
0.60
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0.50
0.40
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0.30
0.20
0.10
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0.00
0
500
1000
Time
1500
Conclusions
Platforms
Metabolite Identification
Metabolite Predictions
Clearance, Safety and
Efficacy
MALDI MS imaging
MALDI-MS
Drug Disposition
Radioactivity
Reactive Metabolites
WIKI
Corporate
p
Knowledge
g
Chromatography/MS
Analytical Knowledge
Bioanalysis & Pharmacokinetics (including modelling, PK/PD and dose
predictions)) can be regarded
p
g
as central to the drug
g discovery
y effort.
Many other platforms, however, can & should be fully used during drug
discovery to ensure the best candidates are selected for drug development.
Key
y References
Additional Information
• All animal studies were ethically reviewed and carried out in accordance with Animals
(Scientific Procedures) Act 1986 and the GSK Policy on the Care
Care, Welfare and Treatment of
Laboratory Animals.
• All human biological samples were sourced ethically and their research use was in accord
with the terms of the informed consent
consent.