1. in the Rat Pup Ultrasonic Vocalization Model
PDE4 Inhibitor Affinity at the High Affinity Rolipram Binding Site (HARBS) Predicts Efficacy
in the Rat Pup Ultrasonic Vocalization Model
F.H . Brucato, A. Hopper, K. Cook, B. Chanas. C. Deng and D.F. Fiorino
1Memory Pharmaceuticals Corp., Montvale, NJ 07645
1. Effect of PDE4 Inhibitors on Pup USV and Locomotion
RESULTS and CONCLUSIONS
1.All PDE4 inhibitors decreased rat pup USVs
suggesting antidepressant potential.
2.The USV and locomotion MED’s varied among the PDE4 inhibitors tested. This suggests the USV assay can discriminate between compounds. Additionally, the USV assay may distinguish between selective antidepressant effects (distress calls) and non-specific effects (locomotion).
3.Of eleven compounds tested:
-USV MEDs ranged from 1 to 100 mg/kg, s.c.
-Brain concentrations at the USV MED ranged from 95 to nM (median = 3173 nM)
-HARBS IC50’s ranged from 5 to 478 nM for 9 compounds. HARBS IC50s were not available for compounds C & D.
4.There was a significant correlation between effective brain concentrations at the USV MED and HARBS affinity (r =0.84), while there was no correlation between effective brain concentrations and in vitro activity at PDE4B or PDE4D. HARBS affinity may predict antidepressant efficacy in the pup USV model.
We would like to thank Patrick Callahan for his assistance and our colleagues at Roche Palo Alto.
INTRODUCTION
There is preclinical and clinical evidence that type-4 phosphodiesterase (PDE4) inhibitors, such as rolipram, have antidepressant properties. PDE4 inhibitors can interact with both low- and high-affinity rolipram binding sites (LARBS and HARBS, respectively), which represent distinct affinity states or conformers. Although the functional significance of LARBS and HARBS is not well understood, recent data suggest that binding to each affinity state mediates a distinct pharmacological effect. In this vein, HARBS may mediate the antidepressant effects of PDE4 inhibitors and antidepressant drugs. Saccomano et al. [1991 J. Med. Chem ] have reported that the antidepressant effects of PDE4 inhibitors correlate with HARBS affinity, as assessed by the tail suspension test in mice and inhibition of [3H]-rolipram binding. In addition, repeated administration of either antidepressant desipramine or fluoxetine, increases HARBS, but not LARBS, in rat cortex and hippocampus. This suggests that different antidepressant classes share a common PDE4 signaling pathway (O’Donnell and Zhang, [2004 TIPS 25(3) ]). In our study, we tested several different PDE4 inhibitors in the pup ultrasonic vocalization (USV) model to evaluate the relationship between antidepressant activity and HARBS.
METHOD
Behavior
Young (d9-11) male and female Sprague-Dawley rat pups were group-housed and raised by their mothers. On experiment day, pups were weighed and separated from their mothers. Thirty minutes after separation, pups were placed individually on a cold stainless steel grid within a Plexiglas chamber. Pre-test performance was measured by number of calls and distance traveled over 5 min. USV’s ranged between 35-45kHz and were collected and digitized using Mini-3 Bat Detectors, Noldus audiofilter and Ultravox software. Animals that emitted fewer than 100 or greater than 900 calls during pre-test, were eliminated from the study. Pups were then injected with vehicle or experimental compound and returned to the test chamber 30 min later for a 5-min test. Results represent the USV or locomotion performance for each dose, compared to that experimental group’s vehicle performance during test (% Control). Data were analyzed using a one-way ANOVA and Dunnett’s post hoc test. *=p<0.05 vs vehicle. Group sizes averaged 8-12 animals/treatment.
Parallel exposure studies were performed to determine blood and brain concentrations at 30 min post-injection for all compounds. For each compound, brain concentration at the USV minimal effective dose (MED) was plotted against the HARBS value.
Enzyme Activity was assessed using standard methods, as reported by Deng et. al. Current Protocols in Pharmacology (2005)
HARBS affinity was determined using the method of Zhao et. al. (2003) JPET 305:
Locomotion
Distress Calls
* * * * * * * * * * * * * *
* * * * * * * * *
% Control
% Control
Drug Dose Drug Dose
(mg/kg) (mg/kg)
2. Effects on USV vs Locomotion Vary Between Compounds
4.HARBS But Not PDE4B or PDE4D IC50 Correlates with Brain Concentration At USV MED
r= 0.84
r=0.077
Normalized Locomotion / Normalized USV
HARBS IC50 (nM)
PDE4D IC50 (nM)
Drug Dose
(mg/kg)
Brain Concentration (nM)
Brain Concentration (nM)
3. PDE4 A/B/D IC 50’s, HARBS, USV MED’s and Brain Concentrations for all Compounds Tested
r=0.070
TEST
MEM A
MEM B
MEM C
MEM D
MEM E
MEM F
MEM G
MEM H
MEM I
MEM J
MEM K
PDE4A IC50 (nM)
117 24
363 *
108
0.9
141 47 96
PDE4B IC50 (nM) 41 11
181 79 10 39
0.4
231 12 26
PDE4D IC50 (nM)
112 7 91 34 56 18
138
HARBS (nM) 13
272
N.A. 16
478 5
7.1 19
298
161
USV MED (mg/kg) 1
100 3 30
Concentration in
Brain at MED (nM)
1184
4748
9941
4285 95
22892
280
1117
1018
3173
8145
B/P Ratio
0.7
0.05
0.07
0.79
0.01
0.69
0.02
0.774
1.76
0.08
0.15
PDE4B IC50 (nM)
Brain Concentration. (nM)