Biology Reference
In-Depth Information
effects, and in patients (4) an antidepressant activity had been demonstrated
(Horowski and Sastre-Y-Hernandez
1985
). The AEs such as nausea and vomiting
prohibited any further development. Looking for the mode of action for cerebral
efficacy, it was found that radioactive rolipram bound to brain structures in vivo
with a dissociation constant which was
100-fold lower than the IC50 for inhibi-
tion of catalytic activity. In binding studies with brain membranes, a dissociation
constant of 1.2 nM was calculated and as binding site a membrane-bound PDE4
was identified. Thus, there was a high affinity and a low affinity rolipram binding
site in the same enzyme (HARBS and LARBS), and the hypothesis was raised that
PDE4 can switch between two conformations which was later corroborated by
evidencing the coexistence of both conformations in one cloned and expressed
enzyme (Torphy et al.
1992a
,
b
).
These experiments indicated that emesis by PDE4 inhibitors might be related to
HARBS. Similarly, with ROS production in neutrophils, we had observed that
rolipram inhibition of release of ROS occurred with an IC50 of 5 nM and was
apparently mediated by HARBS (see Table
7
). The IC50 ratio between catalysis
and ROS release was 42 for rolipram, 6 and 9 for zardaverine and denbufylline, but
for the isochinolines papaverine, benafentrine and tolafentrine the ratio was
<
0.2.
From these results, it was initially assumed that rolipram-mediated decrease in ROS
release from neutrophils was mediated by its interaction with a PDE4 in the high
affinity binding conformation. With the development of tolafentrine, it was deter-
mined that it did only weakly recognise the high affinity binding site of PDE4 and
so differed from rolipram. The interpretation that tolafentrine did only weakly
HARBS was supported by the fact that in clinical studies no emesis was observed
with blood concentrations of up to 5
m
M which exceeded the IC50
<
50-fold.
Similar high blood concentrations (around 1
m
M steady state plasma level after
80 mg infusion) were later achieved in a clinical study with PHT patients (Ghofrani
et al.
2002b
) showing that tolafentrine was the first example of a PDE4 inhibitor
with low emetic potential due to avoidance of interaction with PDE4 in the high
affinity conformation.
>
Table 7 Ratio between inhibition of PDE4 catalysis and ROS release in human neutrophils
Substance IC50 (nmol/l)
PDE4 (PMN) ROS (CL) Ratio
Rolipram 210 5 42
Zardaverine 160 28 6
Denbufylline 100 11 9
Papaverine 110 530 0.21
Benafentrine 4,000 30,200 0.13
Tolafentrine 63 380 0.17
IC50 values for inhibition of PDE4 catalytic activity were determined using enzyme from
chromatographic fractions of a PMN homogenate containing mainly PDE4. IC50 values for
ROS release from neutrophils were calculated from concentration-response curves and chemolu-
minescence related to fMLP-induced oxidative burst was measured. Data are on file at Nycomed
