Richard E. Chipkin

Comparisons between warm and cold water swim stress in mice

The following experiments evaluated the effects of warm- or cold-water swim stress on tail-flick latencies (TFL) in mice. To first determine the appropriate control group, the TFLs of dry-vs-dunked mice were compared. Dry mice had significantly shorter TFLs than dunked mice, implying that the dampness of the mouses tail contributed to the increase in the TFL. Therefore, dunked mice were used as the relevant control for the swum mice. Cold water swimming (2 degrees C) produced a significant increase in the TFL; this was not blocked by the opiate antagonist naloxone (3 mg/kg sc) or potentiated by the enkephalinase inhibitor thiorphan (100 mg/kg sc). Warm water swimming (32 degrees C) up to 3 min produced an inconsistent effect on TFLs, implying that the effects were at the threshold of detectability. Naloxone attenuated and thiorphan modestly potentiated the effects of warm water swimming on TFLs. This suggests that warm water swim stress-induced increases in mouse TFLs may involve opioid pathways, whereas cold water swim stress-induced changes in mice TFLs appear not to be opioid mediated.

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.

Similarity of clozapine and SCH 23390 in reserpinized rats suggests a common mechanism of action

Clozapine at doses up to 100 mg/kg p.o. did not antagonize apomorphine-induced stereotypy in vehicle pre-treated rats. However, if the animals were injected with reserpine (30 mg/kg i.p.) 24 h prior to the test, then clozapine (3-100 mg/kg p.o.) produced a dose-related blockade of apomorphine-induced stereotypy. The blockade of apomorphine-induced stereotypy in reserpinized rats by clozapine was attenuated by the D-2 selective agonist LY 171555 but not the D-1 selective agonist SKF 38393. This profile of agonist reversal of antagonist blockade of apomorphine-induced stereotypy seen with clozapine was identical to that seen with the selective D-1 antagonist SCH 23390. Presumably, the D-2 agonist was active because D-1 receptor systems were inhibited (either at the receptor or at some other post-synaptic site) by clozapine or SCH 23390; this allowed a partial restoration of apomorphine-induced stereotypy via the D-2 system. Therefore, these data indicate that clozapine and SCH 23390 share a common mechanism of action via D-1 receptor systems.

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.

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.

[3H]SCH 39166, A New D1‐Selective Radioligand: In Vitro and In Vivo Binding Analyses

SCH 39166 {(‐)‐trans‐6,7,7a,8,9,13b‐hexahydro‐3‐chloro‐2‐hydroxy‐N‐ methyl‐5H ‐ benzo ‐[d]naphtho[2,1b]azepine} has recently been described as a selective D1 antagonist and has entered clinical trials for the treatment of schizophrenia. The tritiated analogue of this compound, [3H]SCH 39166, has now been synthesized and characterized for its in vitro and in vivo binding profiles. [3H]SCH 39166 binds to D1 receptors in a saturable. high‐affinity fashion, with a KDof 0.79 nM. In competition studies, D1‐selective antagonists like SCH 23390 displaced the binding of [3H]SCH 39166 with nanomolar affinities, whereas antagonists of other receptors exhibited poor affinity. In vivo, [3H]SCH 39166 bound to receptors in rat striatum in a fashion suggestive of D1 selectivity. Further. when the time course for the binding of [3H]SCH 39166 was compared with the behavioral time course of the unlabeled compound, the two durations of action were virtually indistinguishable. Similar studies were performed for SCH 23390 and its tritiated analogue, but the in vivo binding of this radioligand exhibited a duration of action far greater than the behavioral activity of the unlabeled drug. In concert, these data demonstrate that [3H]SCH 39166 selectively labels D1 receptors in vitro and in vivo, and that this drug is superior for in vivo imaging of the D1 receptor.

A characterization of kyotorphin (Tyr-Arg)-induced antinociception

Intracerebroventricular administration of kyotorphin (Tyr-Arg) or Tyr-D-Arg to mice or intrathecal administration of kyotorphin to rats resulted in a dose-dependent, long-lasting, naloxone-reversible analgesia as measured by the 48 degrees C hot plate assay. The potency of kyotorphin was equal to that of Met-enkephalin although its duration of action was substantially longer. Cross-tolerance to kyotorphin could be demonstrated in animals made tolerant to morphine by chronic morphine pellet implantation. Kyotorphin was found to be inactive against column purified enkephalinase A, B and aminopeptidase and indirect evidence would suggest a lack of Met-enkephalin-releasing effect. Thus, kyotorphin represents a unique, naturally occurring peptide with in vivo narcotic-like characteristics and an unknown mechanism of action quite distinct from other opioid peptides.

Studies on the effect of SCH-34826 and thiorphan on [Met5]enkephalin levels and release in rat spinal cord

SCH-34826 and thiorphan are inhibitors of the neutral endopeptidase (NEP; E.C. 3.4.24.11;) that cleaves the opiate peptides [Met5]- and [Leu5]enkephalin at the glycinylphenylalanine bond. These compounds were evaluated for their ability to affect the levels of [Met5]enkephalin-like immunoreactivity (MELI) in the brain and the spinal cord and the release into the extracellular space under resting and K+-evoked conditions. The results showed that oral administration of SCH-34826 (30-100 mg/kg p.o.) or thiorphan (10-30 mg/kg p.o.) had no effect on tissue levels of MELI. In contrast, both agents caused a dose-dependent increase in both the resting and the K+-evoked levels in spinal perfusates, which reached up to 10 times the control values. These data indicate that tissue (presumably intracellular) stores of [Met5]enkephalin are not affected by NEP inhibition and that it is the extracellular effects of the peptide that are potentiated by enzyme blockade. This agrees with the prior results demonstrating that NEP inhibitors require a nociceptive stimulus sufficient to release endogenous stores of [Met5]enkephalins for their actions to be observed.

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.