S C R E E N I N G O F D R U G M E TA B O L I S M T H R O U G H I N T E S T I N A L M I C R O B I OTA
TNO I-SCREEN
INTESTINAL MICROBIOTA SCREENING PLATFORM
FOR DETERMINING METABOLISM OF DRUGS
TNO’s intestinal screening model
(TNO i-screen) helps to quickly
identify pharmacological compounds that are metabolized by
intestinal microbiota. For pharmaceutical companies, searching
for novel pharmaceuticals is a
complex and time-consuming
process. When a novel drug has
been selected, extensive in vitro
and clinical studies are required
to demonstrate its metabolism,
safety and efficacy prior to releasing it to the market. Increasing
evidence has shown that gut
microbiota are involved in the
metabolic transformation of many
drugs, influencing drug pharmacokinetics and thus, efficacy and
safety profiles.
THE TECHNOLOGY EXPLAINED
TNO developed the i-screen platform for
in vitro evaluation of drug metabolism
through intestinal microbiota. Human gut
microbiota are cultured anaerobically in
a multi-well system to mimic the human
intestinal in vivo metabolism. This allows
for determining the potential of intestinal
microbiota to metabolize drug candidates.
The starting population of microbiota in
the TNO i-screen can be a standardized
ex-vivo intestinal microbiota pool
collected from either healthy volunteers,
or for example obese, or lean adults or
even children. This would confer the
inherent differences found in each
individual’s specific gut microflora.
BENEFITS
TNO i-screen enables rapid identification
of drugs that are susceptible to meta­
bolism by intestinal microbiota.
Additionally, it can be used to identify
unknown metabolites transformed by
intestinal microbiota. Normally, human
metabolite profiling and identification
data are generated during Phase II and/
or III of clinical trials. Human metabolites
can thus be identified in preclinical stages,
thereby de-risking and accelerating the
drug development process. Each i-screen
can be simultaneously exposed to a large
number of different conditions, allowing
for cost effective screening of interesting
candidate drugs. Experiments in TNO’s
i-screen are also not subject to lengthy
regulatory processes and allow testing
of drug candidates early on in the
evaluation.
S C R E E N I N G O F D R U G M E TA B O L I S M T H R O U G H I N T E S T I N A L M I C R O B I OTA
EXAMPLES OF THE I-SCREEN AT
WORK
μV
16 - 8 1 37 S E P T E M B E R 2 0 16
μV
220000
120000
36,37
sulfasalazine
200000
110000
100000
180000
5,57
Active metabolite (sulfapyridine)
(>90% of total)
90000
160000
Example 1: Intestinal microbiota driven
80000
140000
70000
metabolism of sulfasalazine
120000
60000
sulfasalazine
100000
TNO has successfully applied the i-screen
(6% of total)
50000
80000
36,30
40000
to demonstrate that sulfa­salazine in the
60000
30000
40000
i-screen system is metabolized as has
20000
20000
10000
been shown in vivo. Sulfasalazine is
0
0
0,00
20,00
40,00
60,00
mins
0,00
20,00
40,00
designed specifically to be metabolized
H+
H+
H
H
H
N
N
N
N
N
Sulfasalazine is metabolized
in TNO’s
by human intestinal microbiota to the
N
N
N i-screen to sulfapyridine
S
S
5-aminosalicylic acid that has therapeutic
O
S
S
N
N
H+
H+
H
H
H
N
N
effect [2]. However, clinical studies
OH
N
O
N
N
N
N
N
S
S
nizatidine
reduced metabolite (m/z 316)
demonstrated that sulfasalazine is mainly
O
S
S
N
N
metabolized to sulfapyridine by the
OH
O
intestinal bacteria, which give rise to
nizatidine
reduced metabolite (m/z 316)
RT: 0.00 - 7.00
side-effects. In this example, microbiota
0.64
2.56
1500000
from healthy adults were exposed to 100
nizatidine
1000000
1.77
0.79 0.89
O
µM sulfasalazine. Samples were taken RT: 0.00 - 7.00
2.41
4.07
500000
0.64
2.56
0.55
2.68
1500000
1.61
1.25
after 0 and 24 hrs. and were analyzed by
3.27 3.66
4.51
2.08
0.50
5.04 5.32 5.51 5.71 6.10 6.39 6.63
0
5.54
0.64
nizatidine
1000000
1500000
HPLC.
1.77
0.79 0.89
O
0.76
uAU
NL:
1.53E6
nm=269.5270.5 PDA
20160518a05
uAU
0
1000000
0.55
0.50
500000
0.64
uAU
500000
60,00 mins
2.41
1.25 1.61
2.08
1.11
4.07
2.68
3.27
3.66
NL:
1.53E6
nm=269.5270.5 PDA
20160518a05
4.51 4.01
5.04 5.32 5.51 5.71 6.10 6.39 6.63
5.54
4.47 4.81
5.92 6.11
3.20 3.34 3.77
5.45
NL:
NL:
1.52E6
nm=269.5270.5 PDA
20160518a06
uAU
uAU
uAU
uAU
uAU
Example 2: Intestinal microbiota driven 1500000
0.56
6.64
1.59
2.50 2.73
2.04
0.46
0.76
0
5.57
metabolism of nizatidine, in collaboration1000000
Reduced
Metabolite
1500000
4.01
1.11
(m/z 316)
500000
with Pfizer Inc.
0.56
1000000
O
4.47
4.81
1.59
3.20 3.34 3.77
5.45
5.92 6.11
6.64
2.73
2.50
2.04
0.46
0.90
0
3.65
In a second example, TNO has success5.57
3.95
500000
1.08
1.50 1.95 2.15
6.64
4.03 4.44 4.78 5.11 5.50
0.53 0.56
2.69
3.18 3.38
5.91 6.24
Reduced
Metabolite
0
fully applied the i-screen in the investiga-1500000
5.61
(m/z 316)
1000000
O
0.90
tion of nizatidine, a pharmaceutical
0.87
3.65
1000000
3.95
500000
1.08
4.78 5.11 5.50
1.50 1.95 2.15
6.64
0.53 0.56
2.69
3.18 3.38
4.03 4.44
5.91 6.24
3.64
compound that is known to be metabo0
500000
1.10
5.61
3.88
1.45 1.72 1.88 2.37 2.64
3.08 3.27
0.53 0.58
4.21 4.41 4.65 5.09 5.41
6.65
5.75 6.06
lized by human intestinal microbiota [3].
0
0.87
1000000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Time
(min)
After the incubation period, the analysis 500000
3.64
1.10
3.88
1.45 1.72 is
3.08 in
3.27TNO’s i-screen
0.53 0.58
2.37 2.64
4.21 4.41 4.65to 5.09
Pfizer
Inc. nizatidine
a reduced
of the samples was performed by Pfizer
5.41
6.65
1.88metabolized
5.75 metabolite
6.06
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Inc. using high resolution mass spectro­
Time (min)
metry. The microbiota from healthy adults
CONTRACTING
were exposed to 50 µM nizatidine and
This service is performed as contract
samples taken after 0, 6, 24 and 48 hrs,
research at TNO. We are always looking
clearly demonstrated the metabolic
for collaborative partners in the next
capacity of gut microbiota.
TNO.NL
pharmaceutical or biotech project.
ADDITIONAL APPLICATIONS
Screening of pro-drugs that are
REFERENCES
transformed to active drugs by
1.Sara JT et al. 1983. Science.
TNO HE ALTHY LIVING
intestinal microbial metabolism
220(4594):325-7.
Diversity by Next Generation
TNO initiates technological and societal
2.Peppercorn MA. 1984. Ann Intern
Sequencing using the MiSeq platform
innovation for healthy living and a dynamic
Med. 101(3): 377-86.
(16S/ITS2 amplicon sequencing)
society.
3.Kandler MP et al. 1986. Drug Metab
(Metagenomic) microbial expression
Dispos. 14(2): 175-82.
profiling using the NextSeq platform
TNO
Metabolic shifts by SCFA analysis
Utrechtseweg 48
(acetate, proprionate, butyrate), BCFA
3700 AJ Zeist
(iso-butyrate, iso-valerate), or numerP +31 (0) 88 866 1649
ous other metabolites for clinical and
The Netherlands
infant nutrition
Steven Erpelinck (BSc. MBA)
[email protected]
1.52E6
nm=269.5270.5 PDA
20160518a06
NL:
1.82E6
nm=269.5270.5 PDA
20160518a07
NL:
1.82E6
nm=269.5270.5 PDA
20160518a07
NL:
1.45E6
nm=269.5270.5 PDA
20160518a08
NL:
1.45E6
nm=269.5270.5 PDA
20160518a08
North America
Tineke Meijers (PhD)
[email protected]
Japan
Kaz Ariga (PhD)
[email protected]