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profiling.
20
These three compounds were evaluated for functional selectivity
against the 5-HT
3
receptor expressed in SHEP1 cells, against endogenous
receptors of TE671 cells to evaluate muscle-like nAChRs (a1b1gd), and against
endogenous receptors of SH-SY5Y cells to evaluate ganglion-like nAChRs
(a3b4). Given the high degree of homology between orthosteric sites in a7
nAChRs and 5-HT
3
receptors,
28
some degree of cross-reactivity was antici-
pated, and a goal was to identify compounds that afforded reasonable binding
selectivity and functional antagonism at the 5-HT
3
receptor. The risk asso-
ciated with 5-HT
3
receptor antagonism was assumed to be minimal and, in fact,
could be beneficial because clinical studies suggest that the potent and selective
5-HT
3
receptor antagonist ondansetron may provide benefits to tardive dys-
kinesia and psychotic symptoms in patients with schizophrenia.
29
In contrast,
compounds that displayed functional 5-HT
3
agonism were not of interest
owing to a potential link to cardiovascular events.
30
Each of the three com-
pounds demonstrated antagonist activity at the 5-HT
3
receptor and a 25-fold
increase in selectivity compared to PNU-282987 in a binding assay for the
a7 nAChR. All three compounds showed no detectable agonist activity
(4100 mM) and negligible antagonist activity at both TE671 and SH-SY5Y cell
receptors. Further, the compounds did not significantly displace [
3
H]cytisine
from rat brain homogenates when examined at a compound concentration of
1 mM, suggesting a high selectivity over the a4b2 nAChR subtype.
One of the key requirements in identifying a compound for clinical devel-
opment was an improved therapeutic index for hERG. In vitro cardiovascular
safety for lead compounds was assessed by measuring their propensity to block
the hERG channel during electrophysiology studies. Prolongation of the QT
interval is believed to correlate with the risk of cardiac arrhythmia in humans
and potentially contribute to ventricular fibrillation.
31
Measuring the effec-
tiveness of a compound to block the hERG potassium channel is an important
preclinical assessment of its proarrhythmic potential.
32
Compounds 6, 8, and 11
were evaluated in a patch-clamp hERG K
1
channel assay at concentrations of
2 and 20 mM,
33
and each showed significantly reduced hERG activity compared
to PNU-282987. To evaluate the interactions with the hERG potassium
channel further, we determined concentration-response profiles for PNU-
282987 and compound 11. In agreement with the screening data, compound 11
was less potent at inhibiting the hERG channel-mediated currents. Although 11
produces insucient blockade at 20 mM, the highest tested concentration to
establish the half-maximal inhibitory concentration (IC
50
), extrapolating the
blockade produced at this concentration (29% for 11) to the fitted curve for
PNU-282987 suggests that the potency for hERG blockade is reduced at least
tenfold (Figure 15.6).
Compounds 6, 8, and 11 were evaluated in a constant-infusion rat in vivo PK
model,
20,26
revealing moderate plasma clearance ranging from 34mLmin
1
kg
1
for furopyridine 11 to 68mLmin
1
kg
1
for benzothiazole 6. Although in
vitro RLM was an accurate predictor of in vivo clearance for compound 11,
RLM under-predicted the in vivo clearance of benzothiazole 6 and indazole 8.
Rat oral bioavailabilities ranged from 65% for furopyridine 11 to 3% for
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