Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 180

Table 14.2 Drug Effects on Rho-HRP Retention in the Brain

Optimal concentration a
( mM) Compound Drug effect (%) P -value

Verapamil 75 57 <0.0001

Phenytoin 100 49 0.0003

Loperamide 1 41 0.0065

RU486 10 17.5 0.0389

Quinidine 100 11 <0.0001

Cyclosporine 10 30 0.0005

Caffeine 12.5 16 0.1007

a Optimal concentration was the concentration that induced the highest effect.

exhibited low fluorescence similar to the level in control DMSO-treated zebrafish,
indicating that this negative compound did not inhibit Pgp efflux.

14.3.4.1 Quantitation of Drug Effects Using Morphometric Image
Analysis To quantitate drug effects on Pgp efflux, we then performed image-
based morphometric analysis in control and drug-treated animals. DMSO control
exhibited 44 19). Due to difficulty in

controlling injection site, volume, and depth, experimental variability was high.
Therefore, in order to compare results, we normalized data by assigning percent
retention in DMSO control in each experiment as baseline, and calculated drug effect
using formula (14.2):

5.3% (mean SE) dye retention ( N ¼

drug effect ð % Þ¼ percent retention ð drug Þ percent retention ð DMSO Þ: ð 14 : 2 Þ

Drug effects are summarized in Table 14.2: verapamil (75 m M), phenytoin (100 m M),

loperamide (10 m M), and cyclosporine (10 m M) caused statistically significant

inhibitory effects; 57% ( P < 0.0001), 50% ( P < 0.0001), 49% ( P ¼ 0.0003),

41% ( P ¼ 0.0065), and 30% ( P ¼ 0.0005), respectively. RU486 (10 m M) and

quinidine (100 m M) exhibited moderate inhibitory effects; 17.5% ( P ¼ 0.0389) and

11% ( P 0.0001), respectively. In comparison, although caffeine, a negative control,

exhibited 16% greater retention than the DMSO control, the difference was not
statistically significant ( P

<

¼ 0.1007), indicating that it is not a Pgp efflux inhibitor.

14.3.4.2 Comparison of Drug Effects in Zebrafish with Results in Cells
andMammals We then compared results in zebrafish with results in cells/tissues
and mammals (Table 14.3). We confirmed that six positive drugs, verapamil,
phenytoin, loperamide, RU486, quinidine, and cyclosporine, inhibited Pgp efflux
in zebrafish brain. Negative control, caffeine, did not induce significant inhibitory
effects. Therefore, the zebrafish brain efflux assay correctly predicted results for all
seven compounds (100%) (Table 14.3).