Development of a Zebrafish Model for Bitter Taste Assessment.

J. Bennett2, A. Coburn1 , A. Coupe2, D. Stedman1,, A. Taylor1, A. Tyler1

1 Pfizer Global R & D, Groton, CT, 06340, USA
2 Pfizer Global R & D, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK

Introduction Fig 4 Effect of concentration on mean food uptake response in zebrafish in initial trial 1
and subsequent repeat trials 2 and 3

Development of simple models to predict bitter taste in humans (paediatrics) is

required to enable improvements in formulation development of paediatric
medicines. A limited number of models are available for evaluating taste e.g. e-
tongue, rat Brief Access Taste Aversion (BATA) model. Although these models
have been shown to have some utility they have a number of limitations and
their application in the drug development process has been limited (1). Juvenile
zebrafish have recently been shown to recognise bitter tastes and further
development of this model could offer a simple alternative to current models
(2,3). In these studies work was undertaken with adult zebrafish (Fig.2) where
feeding was more well established to further understand whether this model
could be used to identify bitter tastes of five compounds with known taste data
in humans.

Materials and Methods

Log doses of test compounds quinine, caffeine, carbamazepine, sildenafil

(1,10,100 mM) and paracetamol (1, 10 and 50 mM) were fed to wild-type
adult zebrafish in a gelatin based food source. Prior to the study zebrafish
were familiarised with eating a gelatin diet in addition to their standard diet. Fig. 5 Effect of concentration on mean food uptake response in zebrafish
The gelatin based food source, Gelly BellyTM(GB), contains a mixture of (combined data from Trials 1,2,3)
vitamins, minerals, krill meal, special mixture of fish and invertebrate feeds,
microalgae and pigment enhancers. The GB was dissolved in hot water,
cooled prior to adding test compounds at concentrations of 1,10,50,100 mM
and allowed to solidify prior to storage at -20C (Fig 3). Each dose of GB was
cut into pieces small enough for fish to eat. Each tank contained five adult
zebrafish (3 females, 2 males) and 3 tanks were used per concentration. The
effect of repeat dosing was examined with zebrafish being given 3 doses
(32.5mg GB + drug) at time 0h, 4h and 24h (Trial 1,2,3). A simple scoring
system was introduced to assess food uptake (Fig.1). Following dosing of the
GB control (no compound) and GB containing test compound fish were
observed for 1 minute and the response scored accordingly.

Fig 1. Scoring System

Scoring System
Observation @ 1 minute Score (Response)
Did not eat GB 0
Ate some of GB 1
Ate all GB 2
Conclusion
Fig 2. Adult Zebrafish
These preliminary studies indicate that adult zebrafish may be a useful model to
identify and evaluate bitter compounds. The presence of quinine, caffeine,
carbamazepine, sildenafil in the food source significantly reduced uptake in a
Fig 3. Drug GB Aliquots
concentration dependent manner. In contrast paracetamol did not impact food
uptake. The data for paracetamol is consistent with human taste panel data where it
was shown to be less bitter than quinine and sildenafil (4).

Acknowledgements
This work was conducted as part of the SPaeDD-UK (Smarter Paediatric Drug
Development-UK) project which is co-funded by Innovate UK and industrial partners:
AZ, Glaxo, Juniper, BMS, Pfizer

Results
Zebrafish showed a dose response effect for caffeine,
carbamazepine, sildenafil with reduced GB consumption at higher
doses and on repeat dosing. The fish consumed less GB on
repeat dosing in trial 2 and 3 relative to the initial dose (Fig.4).
Initial studies with quinine were variable (data not shown) and
optimisation of the dose preparation was undertaken. Subsequent
findings for quinine also showed a dose response effect. In
contrast paracetamol showed no consistent dose response effect
(up to 50mM) Fig. 5 shows the overall dose response data
obtained for all 5 compounds using combined data from trial 1-3.
References
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