No abstract

Cocaine functions, in part, through agonist actions at sigma-1 (σ1 ) receptors, while roles played by sigma-2 (σ2 ) receptors are less established. Attempts to discriminate σ2 receptor-mediated effects of cocaine in locomotor hyperactivity assays have been hampered by the lack of potent and selective antagonists. Certain tetrahydroisoquinolinyl benzamides display high σ2 receptor affinity, and excellent selectivity for binding to σ2 over σ1 receptors. The behavioral properties of this structural class of σ ligands have not yet been investigated. The present study evaluated 5-bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide, 1, a ligand shown by others to bind preferentially to σ2 over σ1 receptors, as well as dopamine D2 and D3 sites. First, we determined binding to monoamine transporters and opioid receptors, and noted 57-fold selectivity for σ2 receptors over the serotonin transporter, and >800-fold selectivity for σ2 receptors over the other sites tested. We then examined 1 in locomotor activity studies using male CD-1® mice, and saw no alteration of basal activity at doses up to 31.6 µmol/kg. Cocaine produced a fivefold increase in locomotor activity, which was attenuated by 66% upon pretreatment of mice with 1 at 31.6 µmol/kg. In vivo radioligand binding studies also were performed, and showed no occupancy of σ1 receptors or the dopamine transporter by 1, or its possible metabolites, at the 31.6 µmol/kg dose. Thus, ligand 1 profiles behaviorally as a σ2 receptor-selective antagonist that is able to counteract cocaine's motor stimulatory effects.

Activation of sigma1 (σ1) receptors contributes to the behavioral and toxic effects of (−)-cocaine. We studied a key step, the ability of (−)-cocaine to occupy σ1 receptors in vivo, using CD-1® mice and the novel radioligand [125I]E-N-1-(3′-iodoallyl)-N′-4-(3″,4″-dimethoxyphenethyl)-piperazine ([125I]E-IA-DM-PE-PIPZE). (−)-Cocaine displayed an ED50 of 68 μmol/kg for inhibition of specific radioligand binding in whole brain, with values between 73 – 80 μmol/kg for heart, lung and spleen. For comparison, an ED50 of 26 μmol/kg for (−)-cocaine occupancy of striatal dopamine transporters (DAT) was determined by inhibition of [125I]3β-(4-iodophenyl)tropan-2β-carboxylic acid isopropyl ester ([125I]RTI-121) binding. A chief finding is the relatively small potency difference between (−)-cocaine occupancy of σ1 receptors and the DAT, although the DAT occupancy is likely underestimated. Interactions of (−)-cocaine with σ1 receptors were assessed further using [125I]E-IA-DM-PE-PIPZE for regional cerebral biodistribution studies and quantitative ex vivo autoradiography of brain sections. (−)-Cocaine binding to cerebral σ1 receptors proved directly proportional to the relative site densities known for the brain regions. Non-radioactive E-IA-DM-PE-PIPZE gave an ED50 of 0.23 μmol/kg for occupancy of cerebral σ1 receptors, and a 3.16 μmol/kg (i.p.) dose attenuated (−)-cocaine induced locomotor hyperactivity by 30%. This effect did not reach statistical significance, but suggests that E-IA-DM-PE-PIPZE is a probable σ1 receptor antagonist. As groundwork for the in vivo studies, we used standard techniques in vitro to determine ligand affinities, site densities and pharmacological profiles for the σ1 and σ2 receptors expressed in CD-1® mouse brain.

Psychostimulant effects of cocaine are mediated partly by agonist actions at sigma-1 (s 1 ) receptors. Selective s 1 receptor antagonists attenuate these effects and provide a potential avenue for pharmacotherapy. However, the selective and high affinity s 1 antagonist PD144418 (1,2,3,6-tetrahydro-5-[3-(4-methylphenyl)-5-isoxazolyl]-1-propylpyridine) has been reported not to inhibit cocaine-induced hyperactivity. To address this apparent paradox, we evaluated aspects of PD144418 binding in vitro, investigated s 1 receptor and dopamine transporter (DAT) occupancy in vivo, and re-examined effects on locomotor activity. PD144418 displayed high affinity for s 1 sites (K i 0.46 nM) and 3596-fold selectivity over s 2 sites (K i 1654 nM) in guinea pig brain membranes. No appreciable affinity was noted for serotonin and norepinephrine transporters (K i .100 mM), and the DAT interaction was weak (K i 9.0 mM). In vivo, PD144418 bound to central and peripheral s 1 sites in mouse, with an ED 50 of 0.22 mmol/kg in whole brain. No DAT occupancy by PD144418 (10.0 mmol/kg) or possible metabolites were observed. At doses that did not affect basal locomotor activity, PD144418 (1, 3.16, and 10 mmol/kg) attenuated cocaine-induced hyperactivity in a dosedependent manner in mice. There was good correlation (r 2 5 0.88) of hyperactivity reduction with increasing cerebral s 1 receptor occupancy. The behavioral ED 50 of 0.79 mmol/kg corresponded to 80% occupancy. Significant s 1 receptor occupancy and the ability to mitigate cocaine's motor stimulatory effects were observed for 16 hours after a single 10.0 mmol/kg dose of PD144418.

This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [3H](+)-pentazocine reached steady state within 6 h at 37 °C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36 ± 0.04 nM; Bmax 967 ± 11 fmol / mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)-N-allylnormetazocine with respect to the (−)-enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [3H]1,3-di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2 min at 25 °C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8 ± 8.3 nM; Bmax 921 ± 228 fmol / mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2 µmol / kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo.

No abstract

Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ1 and σ2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ1 receptor affinities, Ki values of 1.75 nM – 4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ1 receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ1 receptor Ki values in the 20 nM – 30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13 nM – 21 nM range. Most ligands studied exhibited comparable σ1 and σ2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ1 sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ1 Ki = 20.8 nM; σ2 Ki = 16.4 nM) showed over 100-fold higher DAT affinity (Ki = 121 nM) and 6-fold higher SERT affinity (Ki = 128 nM) than the parent SA4503 (DAT Ki = 12650 nM; SERT Ki = 760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions.

Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to μ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [111In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (~99%) and high specific radioactivity (>4000 mCi/μmol). Their log D7.4 values were −2.21 to −1.66. In comparison, DPN is lipophilic, with a log D7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [111In]-labeled DPN-DOTA conjugates for imaging trials.