Michael F. Rafferty

Acute but not chronic activation of the NMDA-coupled glycine receptor with D-cycloserine facilitates learning and retention

The memory-enhancing potential of D-cycloserine (cycloserine) a partial agonist at the glycine recognition site on the NMDA receptor, was evaluated in mice using a thirst-motivated linear maze learning task. Immediate acute post-training injections (10, 20 and 80 mg/kg) significantly improved retention relative to vehicle-injected controls. Retention was also facilitated if cycloserine (3 and 10 mg/kg but not 20 or 40 mg/kg) was administered 20 min before the retention test. Acquisition of the habit was accelerated if cycloserine (3 mg/kg) was injected 20 min before the training session. Acute post-training injections failed to facilitate retention if mice were pretreated with cycloserine (3 mg/kg) b.i.d. for 15 days before training on the maze. These results indicate that acute cycloserine administration can enhance consolidation and retrieval of memory but that desensitization may occur with chronic exposure to the drug.

Antinociceptive effect of spinally delivered prostaglandin E receptor antagonists in the formalin test on the rat

The antinociceptive effect of spinally administered prostaglandin E2 receptor antagonists, SC-51089 and SC-51234A, which are selective for EP1 receptors, was examined in rats using the formalin test. Intrathecal injection of SC-51089 (30-300 micrograms) or SC-51234A (30-300 micrograms) resulted in a significant, dose-dependent, suppression of the second phase (10-60 min), but not the first phase (0-9 min), flinching behavior evoked by formalin injection into the paw. ED25 values and 95% confidence intervals for the second phase were 120 (70-200) micrograms for SC-51089 and 80 (50-140) micrograms for SC-51234A. These data demonstrate that specific nociceptive behaviors are attenuated by spinal prostaglandin E2 receptor antagonists and suggest that prostaglandin E2 is involved in facilitated processing at the spinal level.

[3H]Naltrindole: a potent and selective ligand for labeling δ-opioid receptors

Abstract Naltrindole (NTI) is a selective and potent δ-opioid antagonist which preferentially antagonizes a subset of selective δ-opioid agonists. The purpose of this study was to evaluate whether [ 3 H]NTI, the first radiolabeled δ-opioid antagonist, could selectively label δ-opioid receptors in a synaptosomal preparation. Increasing temperature and protein concentration (0.1–1.6 mg protein) increased the specific binding of [ 3 H]NTI. Monovalent and divalent cations (0.01–100 mM) had minimal effects on the binding properties of [ 3 H]NTI, in contrast to their effects on binding of the delta agonists [ 3 H]DPDPE and [ 3 H]DSLET. Subfractionation of rat brain homogenates revealed that [ 3 H]NTI and [ 3 H]DSLET primarily labeled binding sites in synaptosomal and microsomal fractions, whereas [ 3 H]DPDPE labelled half as many sites in synaptosomal fraction. The B max determined for [ 3 H]NTI in crude synaptosomal fraction was 95 ± 12 fmol/mg. The dissociation constant ( K d ) was determined from three different methods to be 0.08 ± 0.02 nM (Scatchard analysis), 0.07 ± 0.02 nM (competition study) and 0.03 ± 0.005 nM (kinetic analysis). [ 3 H]NTI binding was not significantly inhibited by μ- or κ-opioid ligands or by nonopioid compounds. These results demonstrate that [ 3 H]NTI is a potent and selective radioligand for δ-opioid receptors in rat brain preparations.

Phenylphosphonate monoester analogs of cocaine. Potential haptens for the generation of catalytic antibodies

Abstract A simple method for the stereo-controlled preparation of phenylphosphonate monoesters which are transition state mimics for the hydrolysis of the benzoic acid ester moiety of cocaine is described. Some preliminary results on the use of these mimics for the development of antibodies are also presented.

Quantitative light microscopic localization of [3H]naltrindole binding sites in the rat brain.

The binding of radiolabeled naltrindole ([3H]NTI), a selective delta-opioid antagonist, was characterized using receptor autoradiography. Receptor binding properties were established in brain paste slices which demonstrated one site receptor occupancy with an apparent Kd of 0.25 +/- 0.08 nM (Bmax of 597.5 fmol/mg protein). Autoradiographic localization of [3H]NTI binding sites in the rat brain revealed high densities of these sites in the cortex (layers 1-3 and 6), caudate putamen, accumbens, claustrum, and internal plexiform layer of the olfactory bulb. Moderate to low levels of specific binding were observed in the hippocampus, thalamus, and substantia gelatinosa of the spinal cord.

Antinociception after intracerebroventricular administration of naltrindole in the mouse

Intracerebroventricular (i.c.v.) injection of the delta-opioid receptor antagonist naltrindole hydrochloride (2.2-22.2 nmol) in mice produced a dose-dependent increase in tail flick and hot plate latencies with respective ED50 and 95% confidence limits of 10.6 (8.3-13.9) and 16.4 (9.2-62.3) nmol. This increase in response latencies was antagonized by 1 mg/kg s.c. naloxone or by i.c.v. coadministration of 1.4 nmol ICI-174,864, a selective peptidergic delta-receptor antagonist. Pretreatment 24 h earlier with the irreversible mu-receptor antagonist beta-funaltrexamine (6 nmol i.c.v.) or 1 h earlier with the selective kappa-receptor antagonist nor-binaltorphimine (100 nmol i.c.v.) did not attenuate the antinociceptive effects of naltrindole. These data indicate that high doses of naltrindole may have agonist activity at supraspinal delta-opioid receptors in the mouse.

Acylating phencyclidines irreversibly enhance brain calcium antagonist binding

Phencyclidine was previously shown to allosterically increase the apparent affinity of the dihydropyridine ( [3H]nitrendipine) calcium antagonist binding site in a lysed synaptosomal membrane preparation of rat forebrain. Treatment of a similar preparation of mouse forebrain with 4-isothiocyanato-1-(1-phenylcyclohexyl) piperidine (FOURPHIT), an acylating phencyclidine derivative, resulted in a concentration dependent (0.1-10 microM), irreversible, increase in the apparent affinity of [3H]nitrendipine in contrast to the effects of phencyclidine which were reversible. The FOURPHIT isomer, 1-[1-(3-isothiocyanatophenyl) cyclohexyl] piperidine (METAPHIT), (10 microM) also irreversibly increased the apparent affinity of [3H]nitrendipine, but was much less efficacious than FOURPHIT. Phencyclidine blocked the irreversible increase in the apparent affinity of [3H]nitrendipine produced by FOURPHIT. The interactions of multivalent cations and the calcium antagonist diltiazem with the [3H]nitrendipine binding site were altered following treatment of membranes with FOURPHIT. These studies suggest that FOURPHIT irreversibly interacts with the same sites as PCP, and thus may be a useful tool with which to further probe both the behavioral and biochemical interactions between phencyclidine and the dihydropyridine calcium antagonist binding site.

The N-Boc Group as an Activator for the a-Lithiation of Carbamates: Synthesis of 11-Substituted Dibenzoxazepines

A series of 11-substituted dibenzoxazepines was prepared via α-lithiation utilizing the N-Boc group as an activator. The N-Boc group directs metalation of 5 by n-BuLi to the benzylic C-11 carbon. For 6 which contains 1,3 interrelated directed metalation groups, metalation with n-BuLi occurs at both the 11- and 9- positions. The regioselectivity for the lithiation of 6 can be increased to a ratio of 97:3, by employing LDA as the base, thus providing a convenient and general route to 11-substituted 8-chloro-dibenzoxazepines. By the proper choice of the base, the regioselectivity of lithiation-substitution reactions in the pharmaceutically important heteroaromatic ring systems (5) and (6) can be controlled. The N-Boc group can be readily removed with mild acid treatment