Louis C. Iorio

Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum

A novel benzazepine, SCH 23390, has recently been described as a very potent and selective dopamine D-1 receptor antagonist based on its potent inhibition of dopamine sensitive adenylate cyclase and its selective displacement of 3H-piflutixol from rat striatal receptor sites. In the present study, the in vitro binding of 3H-SCH 23390 to specific striatal receptor sites has been characterized. Binding was saturable and stereospecific, and the results of both saturation and competition studies are consistent with the binding of 3H-SCH 23390 to a single striatal site. A KD of 0.53 nM was obtained through Scatchard analysis. Relative potencies of a variety of neuroleptics in competing with 3H-SCH 23390 and also 3H-spiperone support an interpretation that the single site to which 3H-SCH 23390 binds is the D-1 dopamine receptor. Also, the binding capacity of 3H-SCH 23390 (69 pmoles/gm wet weight) is in agreement with published values for the binding capacities of 3H-piflutixol and 3H-flupentixol. These data, coupled with the low level of non-specific binding encountered with this radioligand (4-8% of total binding at normally employed ligand concentration of 0.3 nM), its high specific activity and its negligible binding to plastic and glass surfaces make it ideally suited for studying interactions with this receptor.

Serotonergic component of SCH 23390: in vitro and in vivo binding analyses

A series of benzazepines related to SCH 23390 were tested for binding to the 5HT-2 receptor. The compounds tested inhibited the binding of 3H-ketanserin with KI values generally greater than those observed for the D-1 receptor, but less than those for the D-2 receptor. When this serotonergic activity was correlated to the D-1 activity, the resulting coefficient was 0.84, indicating a strong correlation between the two activities. Conversely, the 5HT-2 activity did not show a good correlation with the D-2 activity. To further test the significance of the 5HT-2 binding of the SCH 23390, in vivo binding studies were performed using 125I-SCH 38840 in the frontal cortex, an area containing both D-1 and 5HT-2 receptors. The in vivo binding of 125I-SCH 38840 to frontal cortex exhibited peak levels one hour following subcutaneous administration, similar to the time course previously observed in striatum. The binding was both D-1 and tissue specific. Competition studies with selected standards demonstrated that inhibition of the binding to frontal cortex, in contrast to the inhibition observed in the striatum, exhibited a Hill coefficient less than unity, implying interaction at more than one receptor subtype. When SCH 23390 and ketanserin were administered simultaneously, the inhibition of the in vivo binding of 125I-SCH 38840 to striatum was not different than that observed with SCH 23390, alone. However, the inhibition of binding to frontal cortex was significantly greater than that demonstrated with either SCH 23390 or ketanserin, alone, suggesting that 125I-SCH 38840 was binding to both D-1 and 5HT-2 receptors, in vivo.

D1 and D2 dopamine binding site up-regulation and apomorphine-induced stereotypy

Treatments with drugs to up-regulate specific receptors is a strategy often employed in mechanism of action studies. In this type of experiment, changes in the numbers of receptors and concomitant changes in an animals sensitivity to the drug have been used as evidence for the participation of the binding site in the behavior. In these studies, to test for the role of D1 and D2 receptors in apomorphine-induced stereotypy (AIS), dopamine binding sites were up-regulated by appropriate pre-treatments and the ability of these pre-treatments to alter AIS was subsequently investigated. In the first experiment, 19 days of pre-treatment with SCH 23390 or haloperidol selectively increased by 35 and 40% the numbers of striatal D1 and D2 binding sites, respectively, without affecting their affinities. However, when challenged with apomorphine, only the animals pre-treated with the D2 antagonist showed behavioral supersensitivity. In the second experiment, reserpine pre-treatment (30 mg/kg IP, 24-hr pre-test) increased the numbers of D1 binding sites by 18%, but did not significantly alter the numbers of striatal D2 binding sites. Behaviorally, these rats were supersensitive to apomorphines stereotypy-inducing effects; however, they also showed an increased sensitivity to the ability of either haloperidol or SCH 23390 to block AIS. Moreover, this blockade was only attenuated by a D2 (but not a D1) agonist. Collectively, these data suggest that AIS is mediated by both D1 and D2 binding sites, but that D2 binding sites have a more important role.

Potentiation of stress-induced analgesia (SIA) by thiorphan and its block by naloxone

Exposure of rats to inescapable footshock produces an analgesic effect. To determine if endogenously released enkephalins play a role in this phenomenom, rats were treated with the enkephalinase inhibitor thiorphan (T), exposed to inescapable stress, and tested in the tail-flick test for antinociception. T (10-100 mg/kg sc) caused a dose-related potentiation of both the peak effect and the duration of the SIA. This effect was blocked by doses of naloxone (1 mg/kg sc) that did not affect baseline response latencies.

Selective affinity of 1-N-trifluoroethyl benzoidiazepines for cerebellar type 1 receptor sites

In binding studies with rat brain membranes, 1,4-benzodiazepines containing a trifluoroethyl moeity at the 1-N position, including halazepam and quazepam, had significantly higher affinities for binding sites in cerebellum than in cortex. This selectivity for cerebellar sites is not a property of benzodiazepines without the trifluoroethyl moiety, but is similar to that seen with the triazolopyridazines. Since halazepam and quazepam, like the triazolopyridazines, have behavioral effects in animals at doses much lower than those that cause ataxia, it is tempting to attribute this separation of pharmacologic activities to differential activity at subpopulations of benzodiazepine receptors. Further work is necessary to clarify this possibility.

Potentiation of [D-ala2]enkephalinamide analgesia in rats by thiorphan.

The effect of thiorphan, an inhibitor of enkephalin dipeptidyl carboxypeptidase, was tested in the rat tail-flick test. When given alone up to 100 mg/kg s.c., thiorphan had no effect. However, thiorphan (30 mg/kg, s.c.) potentiated intraventricularly administered [D-Ala2,Met5]enkephalinamide (DEAEM) and its [Leu5]-derivative, whereas it had no effect on [D-Ala2,Met5]enkephalin, [D-Met2,Pro5]enkephalinamide, or morphine. Potentiation of DAEAM by thiorphan was dose-responsive and naloxone reversible. The most efficient enhancement of the analgesic effects of DAEAM was seen when the two drugs were co-administrated in rapid succession. Thus, thiorphan potentiation of opioid-peptide induced analgesia in the rat is similar to that seen in the mouse.

Benzazepines: Structure-Activity Relationships Between D1 Receptor Blockade and Selected Pharmacological Effects

SCH 23390 is (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1-H-3-benzazepine. Its synthesis was based on the benzazepine series synthesized more than 15 years ago by Dr. Lewis Walter and Mr. Wei Chang at Schering Research Laboratories, and highlighted by the work done with the 7,8-dimethoxy analog of SCH 23390, SCH 12679. This earlier-described drug (Barnett et al., 1974) manifested potent inhibition of aggression invoked in animals by such diverse methods as social isolation, septal and olfactory bulb lesions and electrical stimulation, and aggression occurring spontaneously in monkeys (attack phenomena) and rats (muricide). With respect to antipsychotic potential, SCH 12679 did differ from the standards in that it did not block conditioned avoidance responding (CAR) in rats, an effect which we consider an important index of antipsychotic potential.

Characterization of the radioiodinated analogue of SCH 23390: In vitro and in vivo D-1 dopamine receptor binding studies

A new radioiodinated molecule, 125I-SCH 38840 (previously referred to as 125I-SCH 23982), has been recently reported to be a D-1 dopamine receptor ligand. The current study confirms and expands the characterization of both the radiolabeled and unlabeled forms of this compound, as well as describing the development of an in vivo D-1 receptor binding assay utilizing the 125I-SCH 38840. The binding of 125I-SCH 38840 to rat striatal membranes, in vitro, was saturable and exhibited a KD of 1.47 nM. Competition studies using 125I-SCH 38840 exhibited a pharmacological profile consistent with the proposal that 125I-SCH 38840 was binding to the D-1 receptor. Further studies with the unlabeled SCH 38840 demonstrated that it inhibited dopamine-stimulated adenylate cyclase with a KI of 66.1 nM, indicating that SCH 38840 was acting as a D-1 antagonist. Behavioral studies demonstrated that SCH 38840 (MED = 1.0 mg/kg, s.c.) blocked conditioned avoidance responding in rats, a measurement considered predictive of anti-psychotic activity in man. In vivo binding of 125I-SCH 38840 to rat striatum following s.c. administration was specific. Peak striatal levels were observed 1 h after injection, with measurable binding observed out to 8 h post-treatment. The displacement of the in vivo binding by unlabeled standards again suggested a D-1 selective interaction. The half-life of the in vivo binding of 125I-SCH 38840 was approximately 1.25 h, and was nearly equivalent to the half-life of the anti-CAR activity of unlabeled SCH 38840. These results clearly demonstrate the D-1 nature of SCH 38840s behavioral activity and strengthen the anti-psychotic potential of a D-1 antagonist.

Selective affinity of the benzodiazepines quazepam and 2-oxo-quazepam for BZ1 binding site and demonstration of 3H-2-oxo-quazepam as a BZ1 selective radioligand

Quazepam and 2-oxo-quazepam are novel benzodiazepines containing a trifluoroethyl substituent on the ring nitrogen at position #1. Detailed competition binding experiments (25 to 30 concs.) at 4 degrees C were undertaken with these compounds versus 3H-flunitrazepam using synaptic membranes from rat cortex or cerebellum. Unlike other benzodiazepines, both quazepam and 2-oxo-quazepam distinguished two populations of 3H-flunitrazepam binding sites in rat cortex which were present in roughly equal proportions and for which the compounds displayed a greater than 20-fold difference in affinity. In cerebellum, no such discrimination of sites was noted for 2-oxo-quazepam, but quazepam did distinguish a small, low affinity (15% of total) population of sites. 3H-2-oxo-quazepam was prepared and used in competition studies to substantiate the conclusion that these compounds discriminate two populations of benzodiazepine sites in rat cortex. This new radioligand was shown to specifically label BZ binding sites with high affinity in a saturable manner. The competition experiments were then conducted using 3H-2-oxo-quazepam at a radioligand concentration sufficiently low (0.5 nM) to ensure that only the higher affinity binding sites which 2-oxo-quazepam discriminates would be occupied. Competition experiments in both cortex and cerebellum under these conditions indicated single site binding for unlabelled quazepam and 2-oxo-quazepam in every instance. This suggests that 3H-2-oxo-quazepam should be a useful new tool for selectively labeling and studying the BZ1 population of benzodiazepine binding sites.

Sleep-inducing effects of three hypnotics in a new model of insomnia in rats

Sleep-inducing effects of hypnotic drugs are difficult to demonstrate in rats because of high baseline sleep times. Most increases in slow wave sleep (SWS) following the administration of hypnotics have been found to be at the expense of REM sleep rather than waking. In the first experiment we found that rats chronically implanted with electrodes for recording ECoG and EMG slept significantly less during the first two hours of the dark period when housed under 16-hr light 8-hr dark (16L/8D) than when housed under 12L/12D conditions. The second experiment examined the effects of flurazepam, phenobarbital and thalidomide administered orally at the onset of the two-hour period of increased waking resulting from the 16L/8D lighting. All three drugs caused dose-dependent reductions in waking and increases in SWS with no alterations in REM% of total sleep time. Sleep onset latency was also significantly was also significantly reduced by all three drugs. As in man, flurazepam was the most potent, and thalidomide was the least potent of the hypnotics in rats.