Michael P. Johnson

Effects of enantiomers of MDA, MDMA and related analogues on [3H]serotonin and [3H]dopamine release from superfused rat brain slices

The primary amines 3,4-methylenedioxyamphetamine (MDA), and 1-(1,3-benzodioxol-5-yl)-2-butanamine (BDB) were measured for efficacy in release of [3H]serotonin (5-HT) from rat hippocampal slices, and release of [3H]dopamine (DA) from rat caudate nucleus slices. The N-methyl derivatives of MDA and BDB, MDMA and MBDB, respectively, and the optical antipodes of these four agents were compared in this paradigm. All of the test compounds demonstrated a similar efficacy of [3H]5-HT release in the micromolar concentration range. No significant stereoselectivity was seen in measurements of 5-HT release. However, striking differences were found between the test compounds when [3H]DA release was studied. N-methylation of racemic MDA resulted in a decreased ability to release DA, while side chain extension from alpha-methyl to alpha-ethyl completely abolished this activity. Stereoselectivity for the S-(+)-isomers of MDA and MDMA was also demonstrated in the DA release studies. Correlation of these biochemical findings with human subjective reports indicates that serotonin release may play a more important role in the mechanism of action than does dopamine release.

Six-Hour Window of Opportunity for Calpain Inhibition in Focal Cerebral Ischemia in Rats

BACKGROUND AND PURPOSE Stroke patients often experience a significant temporal delay between the onset of ischemia and the time to initiation of therapy. Thus, there is a need for neuroprotectants with a long therapeutic window of opportunity. The efficacy of a potent, central nervous system-penetrating calpain inhibitor (MDL 28,170) was evaluated in a temporary model of focal cerebral ischemia to determine the window of opportunity for intracellular protease inhibition. METHODS An ex vivo brain protease inhibition assay established pharmacodynamic dosing parameters for MDL 28,170. Middle cerebral artery (MCA) occlusion was accomplished by advancing a monofilament through the internal carotid artery to the origin of the MCA. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium-stained brain sections. RESULTS Maximal inhibition of brain protease activity was observed 30 minutes after injection of MDL 28,170 with an estimated pharmacodynamic half-life of 2 hours. MDL 28,170 caused a dose-dependent reduction in infarct volume when administered 30 minutes after MCA occlusion. A window of opportunity study was conducted to determine the maximal delay between the onset of ischemia and the initiation of efficacious therapy. MDL 28,170 reduced infarct volume when therapy was delayed for 0.5, 3, 4, and 6 hours after the initiation of ischemia. The protective effect of MDL 28,170 was lost after an 8-hour delay. CONCLUSIONS These data indicate that the therapeutic window of opportunity for calpain inhibition is at least 6 hours in a reversible focal cerebral ischemia model. This protection is observed despite the lethal hypoxic and excitotoxic challenge, suggesting that calpain activation may be an obligatory, downstream event in the ischemic cell death cascade.

Metabotropic glutamate 2 receptor potentiators: receptor modulation, frequency-dependent synaptic activity, and efficacy in preclinical anxiety and psychosis model(s)

RationaleTo increase subtype selectivity and provide a novel means to alter receptor function, we discovered and characterization potentiators for the metabotropic glutamate 2 receptor (mGlu2).Methods and resultsA class of 3-pyridylmethylsulfonamides (e.g., 3-MPPTS; 2,2,2-trifluoro-N-[3-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) were found to be potent, subtype-selective potentiators of human and rat mGlu2. The sulfonamides increased agonist potency in functional assays but did not displace orthosteric radiolabeled antagonist or agonist binding to cloned mGlu2 receptors. Rather, the modulators increased the affinity of most of the orthosteric agonists including glutamate, DCG-IV (2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)glycine), and LY354740 (1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate). In striatal brain slices, LY354740 inhibited evoked excitatory postsynaptic potentials (EPSPs) equally well following either a low- (0.06 Hz) or high (4 Hz)-frequency stimulation of corticostriatal afferents. In contrast, the mGlu2 potentiator cyPPTS (2,2,2-trifluoro-N-[3-(cyclopentyloxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) inhibited striatal EPSPs only at higher frequencies of stimulation (2 and 4 Hz). Several sulfonamides including 4-MPPTS, 4-APPES (N-[4-(4-carboxamidophenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride monohydrate) and/or CBiPES N-[4′-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride) were tested in mGlu2/3 agonist-sensitive rodent model(s) of anxiety and psychosis. As seen with LY354740, both 4-MPPTS and 4-APPES were efficacious in a rat fear-potentiated startle paradigm. Likewise in mice, CBiPES attenuated a stress-induced hyperthermia and PCP-induced hyperlocomotor activity. Furthermore, CBiPES mediated alteration in PCP-induced hyperlocomotor activity was sensitive to mGlu2/3 antagonist pretreatment.ConclusionsTaken together, the data indicate mGlu2 receptor potentiators have a unique use-dependent effect on presynaptic glutamate release, and show efficacy in several mGlu2/3-sensitive animal models of psychiatric disorders.

[3H]Monoamine releasing and uptake inhibition properties of 3,4-methylenedioxymethamphetaimne and p-chloroamphetamine analogues

The ability of several 3,4-methylenedioxymethamphetamine (MDMA) analogues to inhibit the uptake of [3H]serotonin (5-HT), dopamine (DA) and norepinephrine (NE) into synaptosomes was examined. In addition, the ability of the compounds to inhibit the uptake of [3H]5-HT and DA into synaptosomes from rats pretreated with reserpine (5 mg/kg i.p., 16 h pretreatment) was compared to control experiments. All of the test compounds were found to be potent releasers of non-vesicular 5-HT (the reserpine IC50 was significantly smaller than the control IC50). The range of 5-HT inhibitory activity corresponds well to the small range of ED50 values of the test compounds to substitute in drug discrimination experiments with animals trained to discriminate MDMA or S-(+)-N-methyl-1-(1,3-benzodioxol-5-yl)-2-aminobutane (S-MBDB) from saline. In contrast, there was a wide range of potency for the inhibition of NE and DA uptake. In addition, several of the analogues appeared to be pure uptake inhibitors of DA while others were found to be releasers of non-vesicular DA. Several of the compounds were very selective for 5-HT over DA or NE uptake inhibition, including 3-methoxy-4-methylamphetamine (MMA) and 5-methoxy-6-methyl-2-aminoindan (MMAI). A correlation was noted between the 5-HT neurotoxic potential of some of the test compounds and their relative ability to induce a release of non-vesicular DA. The potential catechol metabolites of the methylenedioxy-substituted compounds also showed potent monoamine releasing properties, suggesting that metabolism may play a role in the neurotoxic actions of some of these drugs. The present data support the hypothesis that drug-stimulated non-vesicular 5-HT release is primarily responsible for the discriminative cue of MDMA.

AMPHETAMINE DERIVATIVES INDUCE LOCOMOTOR HYPERACTIVITY BY ACTING AS INDIRECT SEROTONIN AGONISTS

Derivatives of amphetamine are potent releasers of both dopamine (DA) and serotonin (5-HT), but the relative contributions of DA and 5-HT release to the behavioral effects of these drugs have not been established. Previously, S-(+)3,4-methylendioxymethamphetamine (S-(+)MDMA) was found to produce locomotor hyperactivity in rats which was dependent on 5-HT release. The present study found that MBDB (1.25, 2.5, 5.0 or 10.0 mg/kg), the alpha-ethyl derivative of MDMA that produces little or no direct DA release, also induced locomotor hyperactivity that lasted for greater than 60 min after the 5.0 and 10.0 mg/kg doses. MBDB produced spatial patterns of locomotor hyperactivity and suppression of exploratory activity (holepokes and rearings) very similar to the behavioral syndrome produced by MDMA. MBDB-induced hyperactivity was blocked by pretreatment with the selective 5-HT uptake inhibitor fluoxetine (2.5 or 10 mg/kg), suggesting that MBDB produced behavioral effects via uptake-carrier mediated release of 5-HT. Similarly, fluoxetine pretreatment blocked the locomotor hyperactivity produced byS-(+)3,4-methylenedioxyamphetamine (3.0 mg/kg) orp-chloroamphetamine (2.5 mg/kg), supporting a serotonergic basis for the action of these drugs. Tissue levels of 5-HT and its metabolite 5-HIAA were decreased 40 min after administration ofS-(+)MDMA (3.0 mg/kg) or MBDB (5.0 mg/kg), and these decreases were prevented by fluoxetine pretreatment.S-(+)MDMA also produced a fluoxetine-sensitive increase of tissue DA levels, suggesting that 5-HT release may indirectly result in increased DA release, although MBDB did not significantly increase DA levels. These results point to a central role for 5-HT release in the stimulant-like behavioral effects of substituted derivatives of amphetamine.

Comparative serotonin neurotoxicity of the stereoisomers of fenfluramine and norfenfluramine

The optical isomers of fenfluramine and norfenfluramine were administered to rats to examine their relative potency for destruction of serotonin neurons. Rats were sacrificed one week following a single 10 mg/kg SC injection of one of the four compounds and monoamine and metabolite levels in the frontal cortex and hippocampus brain regions were examined by HPLC-EC techniques. In addition, [3H]-paroxetine binding to homogenates of these brain regions was determined. With the exception of hippocampal 5-HT levels following d-fenfluramine treatment, there was a decrease in all the serotonergic markers assayed, following treatment with the d-enantiomers of fenfluramine and norfenfluramine. No decrease in any serotonergic marker was seen at this dose following treatment with the l-enantiomers of fenfluramine or norfenfluramine. Also, none of the drug treatments resulted in a significant decrease in catecholamines or their metabolites. With all the serotonergic markers examined, d-norfenfluramine was found to cause a significantly greater decrease than d-fenfluramine. The significance of these results is discussed in terms of the hypothesis that the long-term serotonergic deficits observed with d-fenfluramine may result from its metabolite, d-norfenfluramine.

[125I]-2-(2,5-Dimethoxy-4-Iodophenyl) aminoethane ([125I]-2C-I) as a label for the 5-HT2 receptor in rat frontal cortex

Recent studies of 5-HT2 receptor binding have involved the use of radiolabeled agonists. This report describes the use of [125I]-2-(2,5-dimethoxy-4-iodophenyl)aminoethane ([125I]-2C-I) as a label for low-density 5-HT2 agonist binding sites. A nonhydrolyzable analog of GTP, GppNHp, was found to inhibit the high affinity binding of [125I]-2C-I. 5-HT and several 5-HT2 agonists and antagonists displayed high affinity for this site. In addition, a significant decrease in the Bmax value, but not the KD for [125I]-2C-I was observed at 37 degrees C as compared to that observed at 24 degrees C. Several structure-activity relationships were investigated for displacement of [125I]-2C-I, and the results are consistent with the importance of this receptor in the mechanism of action of hallucinogens. This study demonstrates the utility of [125I]-2C-I as a novel radioligand and provides further data that the 5-HT2 receptor is significantly linked to hallucinogenic activity for several compounds.

Behavioral, biochemical and neurotoxicological actions of the α-ethyl homologue of p-chloroamphetamine

The present set of experiments was designed to examine the effects of extension of the alpha-methyl of p-chloroamphetamine (PCA) to an alpha-ethyl. Therefore, the alpha-ethyl homologue of PCA, 1-(4-chlorophenyl)-2-aminobutane (CAB), was compared to PCA in a number of pharmacological assays. CAB was 2-fold less potent than PCA at inhibiting synaptosomal uptake of [3H]5-hydroxytryptamine ([3H]5-HT), and 5-fold less potent at inhibiting uptake of [3H]dopamine ([3H]DA). In drug discrimination assays, CAB was approximately 3-fold less potent than PCA in animals trained to discriminate 3,4-methylenedioxymethamphetamine (MDMA) or its alpha-ethyl homologue, S-(+)-N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (S-(+)-MBDB), from saline. Monitoring with in vivo microdialysis, 10 mg/kg of PCA caused a large increase in extracellular DA and a significant decrease in 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum. In contrast, 11 mg/kg CAB caused no increase and 22 mg/kg CAB caused only a slight increase in extracellular DA. Both doses of CAB caused a decrease in extracellular DOPAC. The potential 5-HT neurotoxicity of CAB was examined by measuring monoamine and metabolite levels and [3H]paroxetine binding at one week following acute doses. A 10 mg/kg dose of PCA caused an 80% decrease in cortical and hippocampal serotonergic markers, while an equimolar dose of CAB decreased only hippocampal 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels. However, 22 mg/kg of CAB produced a 20-40% decrease in all serotonergic markers. Thus, extension of the alpha-alkyl significantly decreases the dopaminergic effects of PCA. The similar decrease in relative 5-HT neurotoxicity and the decreased ability to alter dopaminergic systems in vivo and in vitro supports the involvement of DA in the neurotoxicity of PCA.

Serotonin Neurotoxicity in Rats After Combined Treatment With a Dopaminergic Agent Followed by a Nonneurotoxic 3,4-Methylenedioxymethamphetamine (MDMA) Analogue

There is increasing evidence linking dopamine (DA) to the long-term serotonergic (5-HT) neurotoxic effects of certain substituted amphetamines such as 3,4-methylenedioxymethamphetamine (MDMA). The present study was undertaken to examine the importance of DA metabolism, uptake inhibition and release in the long-term effects of these drugs by combining various dopaminergic agents with an analogue of MDMA that had low neurotoxic liability, namely 5,6-methylenedioxy-2-aminoindan (MDAI). Monoamine and metabolite levels and the number of 5-HT uptake sites (using [3H]paroxetine binding) were determined 3 hours or 1 week after treatments. Combining the monoamine oxidase inhibitors, clorgyline (MAOA selective) or deprenyl (MAOB selective) with MDAI did not result in any long-term reductions of serotonergic markers. Similarly, combining the DA uptake inhibitor GBR-12909 with MDAI did not result in any long-term changes in monoamine levels at 1 week. In contrast, a single pretreatment of posttreatment with the nonvesicular DA releaser S-amphetamine and MDAI resulted in small but significant long-term changes in monoamine levels. More importantly, if a subacute dosing regimen (every 12 hours for 4 days) was utilized, the combination of S-amphetamine with MDAI resulted in a marked long-term decrease in the levels of cortical, hippocampal and striatal 5-HT, 5-HIAA and the number of 5-HT uptake sites. The results are discussed in terms of the significance of DA and especially nonvesicular DA release in the long-term effects of MDMA-like drugs.

Synthesis and Pharmacological Characterization of C4-Disubstituted Analogs of 1S,2S,5R,6S-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: Identification of a Potent, Selective Metabotropic Glutamate Receptor Agonist and Determination of Agonist-Bound Human mGlu2 and mGlu3 Amino Terminal Domain Structures.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4β-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.