Frank HJ Schuren1
R Scott Obach2
Loretta M. Cox 2
B Usta1
M. Heerikhuizen1
H. Rahaoui1
Irene HG Nooijen1
Steven Erpelinck1
Wouter HJ Vaes1
Evita van de Steeg1

CONVERSION OF DRUG METABOLITESBACK TO PARENT DRUGS BY HUMAN GUTMICROBIOTA IN AN EX VIVOFERMENTATION SCREENING PLATFORM
Visit us at booth #21

P69

1TNO, Human Biology & Microbiology, Healthy Living,

The Netherlands
2 Pfizer, Inc., Groton, CT, USA

Evita.vandesteeg@tno.nl

INTRODUCTION➢ Human gut microbiota play an important role indrug kinetics and metabolism by activating,inactivating, or even toxifying orally administereddrugs.
➢ Metabolism of selected drugs by pooled microbiota
in the ex vivo fermentation platform has beenshow before (van de Steeg et al., 2018), butconversion of metabolites back to parent (as partof enterohepatic circulation) is currently not highly
studied.

ARESULTSB

p o o le d t = 0➢ Previously we have shown that out of 12 parent
drugs 5 were metabolized in i-screen
experiments and specific reduction metabolites
could be shown (microbiota-induced metabolism
D
p o o le d t = 2 4
was demonstrated for risperidone, sulindac,sulfinpyrazone, nizatidine and sulfasalazine).2➢ Here we have analyzed the capability of adulthuman colon microbiota to metabolize drug
metabolites generated in the liver back to theirrespective parent drugs, since these drug
metabolites may through biliary excretion end up
in the GI–tract in vivo.

C

p o o le d t = 0

p o o le d t = 0

A

p o o le d t = 0

B

p o o le d t = 0C

p o o le d t = 0D

p o o le d t = 2 4Ep o o le d t = 2 4F

p o o le d t = 2 4Ep o o le d t = 2 4F

B a c te r o id e te s

p o o le d t = 2 4

F ir m ic u te s
A c tin o b a c te r ia
P r o te o b a c te r ia
u n c la s s ifie d _ B a c te r ia

B a c te r o id e te s
F ir m ic u te s
A c tin o b a c te r ia
P r o te o b a c te r ia
u n c la s s ifie d _ B a c te r ia

Figure 2. Microbial composition of pooled human colon microbiota
before (t=0) and after ex vivo fermentation in i-screen for 24 hours(t=24). Intra-experimental variation is shown by presenting A, B
and C and D, E and F representing triplicate incubations of one
experiment. Data are presented as mean relative abundance of theindividual microbial species (n=3).

Table 1. Overview of metabolic faith of 8 Investigated Drug metabolites Susceptible to Microbial Metabolism in I-screen.
Substrate(metabolite)

Product(parent drug)

Reaction TypeObservation

Acetaminophen sulfate

Acetaminophen

Mycophenolic Acid
Glucuronide

Mycophenolic Acid

Sertraline-N-Carbamoyl
Glucuronide

Sertraline

Benzydamine N-Oxide

Benzydamine

Imipramine N-Oxide

Imipramine

SN-38 Glucuronide

SN-38

Raloxifene 4’-Glucuronide

Raloxifene

Raloxifene 6-Glucuronide

Raloxifene•••
Acyl glucuronide hydrolysis•
Carbamoyl glucuronide hydrolysis ••••N-Oxide Reduction•••
N-Oxide Reduction•••
Phenol Glucuronide Hydrolysis••
Phenol Glucuronide Hydrolysis••
Phenol Glucuronide Hydrolysis•

Reaction was complete at 6hNo reaction in microbiota free incubation
Reaction occurs in 6h and continued over 24h
No reaction in microbiota free incubation
Very fast reaction, within minutes after incubation
Reaction was complete after 6h
Sertraline continued to decline in gut microbiota between 6 and 24h
Reaction was complete after 6hSome conversion to parent in absence of gut microbiota suggesting instability
Benzydamine continued to decline in gut microbiota between 6 and 24h
Reaction was complete after 6hSome conversion to parent in absence of gut microbiota suggesting instability
Imipramine was further metabolized by gut microbiota between 6 and 24h
Reaction occurs in 6 h and continued over 24h
No reaction in microbiota free incubation
Reaction occurs in 6 h and continued over 24h
No reaction in microbiota free incubation
Reaction occurs in 6 h and continued over 24h
No reaction in microbiota free incubation

AIMAB
60

25 µM SN38G50 µM SN38G40

2000

Figure 1. Graphical abstract of ex vivo fermentation platform to
study deconjugation of drug metabolites by the human microbiome.

METHODS➢ A set of 8 drug metabolites (acetaminophensulphate,
mycophenolicacid
glucuronide,
sertraline-N-CO-glucuronide,
benzydamine Noxide, imipramine N-oxide, SN38 glucuronide(SN38G), raloxifene 4’-glucuronide and raloxifene
6’-glucuronide) was incubated for 6 and 24 hours
with human colonic microbiota (pool of 7 healthy
adult fecal samples) under strictly anaerobic
conditions [1,2].
➢ In order to determine the kinetics of the metabolic
conversion, SN38G was applied as a model drug.
Linearity of the metabolic conversion was studied
over 6h, and kinetics of the metabolic conversion
was studied at multiple concentrations of SN38G(5, 25, 50 and 100 µM). All samples were analyzed
using high-performance liquid chromatograph.

REFERENCES
1 Ladirat SE et al. J. Micr. Methods (2013) 92: 387-397
2 Van de Steeg E et al. Drug Metab Dispos (2018) 46:

1596-1607

umol/ug bacterial DNA/hPercentage SN38 formation➢ To study and quantify the fate of drug metabolites(generated in the liver) upon exposure to gut
microbiota in the i-screen platform, a translational
ex vivo fermentation platform simulating thehuman colonic microbiota conditions.

Phenol sulfate hydrolysis

246Time (h)8300200100
Vmax: 333 µmol/µg bacterial DNA/h (95% CI: 285-403)
Km: 28 µM (95% CI: 17-45).

Figure 3. (A) Metabolic fate of SN38
glucuronide in ex vivo fermentation
platform over time (applying 10-folddilutedmicrobiome
stocks,(B)Concentration dependent metabolic
conversion of SN38G to SN38 by humanmicrobiota (3h of incubation with 10fold diluted microbiome stocks).

0050

100

150µmol (SN38G)

CONCLUSIONS➢ Enterohepatic circulation (EHC) of drugs involves the following processes: metabolite formation(generally in the liver), secretion into the bile, then back-converted in the GI-tract and (potentially)
re-absorbed.
➢ All tested drug metabolites showed microbiota-based conversion to parent drug within 6h,
demonstrating different reaction types including phenol sulfate hydrolysis, phenol glucuronidehydrolysis, acyl glucuronide hydrolysis, carbamoyl glucuronide hydrolysis, and N-oxide reduction.
➢ Deconjugation of SN38G into SN38 was linear up to 4h of incubation, with an average Vmax of 333µmol/µg bacterial DNA/h (95% confidence interval (CI): 285-403) and average Km of 28 µM (95% CI:17-45).
➢ The i-screen platform can serve as an in vitro system to quantify metabolic conversion of drugs bygut microbiota.

FUTURE PERSPECTIVES➢ Investigating inter-individual differences in colonic metabolism (personalized medicine).
➢ Modulation of the microbiome to influence these metabolic processes offers interesting newtreatment options.
➢ Combining data from different in vitro and ex vivo platforms to predict and model EHC byphysiologically-based pharmacokinetic modeling.