The sigma receptor ligand N-phenylpropyl-N'-(4-methoxyphenethyl)3piperazine (YZ-067) enhances the cocaine conditioned-rewarding properties while inhibiting the development of sensitization of cocaine in mice.

RATIONALE: The N-phenylpropyl-N'-substituted piperazines SA-4503 (N-phenylpropyl-N'-(3,4-dimethoxyphenethyl)piperazine) and YZ-185 (N-phenylpropyl-N'-(3-methoxyphenethyl)piperazine) bind to sigma (σ) receptors and block the development of cocaine-induced conditioned place preference at concentrations that inhibit cocaine-induced hyperactivity. YZ-067 (N-phenylpropyl-N'-(4-methoxyphenethyl)piperazine) also binds to sigma receptors and attenuates cocaine-induced hyperactivity in mice. OBJECTIVES: The present study determined the effect of YZ-067 on the development and expression of cocaine (66 µmol/kg or 33 µmol/kg) conditioned place preference (CPP) and locomotor sensitization in mice. RESULTS: YZ-067 (10 or 31.6 µmol/kg) did not have intrinsic effects on place preference or place aversion. Interestingly, the 31.6 µmol/kg YZ-067 dose enhanced the development of cocaine place preference, while 10 µmol/kg YZ-067 attenuated the development of cocaine-induced locomotor sensitization. However, YZ-067 did not alter the expression of cocaine place preference nor cocaine-induced locomotor sensitization. In follow-up studies, YZ-067 did not affect performance in the zero maze or rotarod, indicating that sigma receptors probed by this ligand do not regulate anxiety-like or coordinated motor skill behaviors, respectively. CONCLUSION: Overall, these results are consistent with previous studies demonstrating a role for sigma receptors in the behavioral effects of cocaine. However, the present findings also indicate that N-phenylpropyl-N'-substituted piperazines do not strictly block cocaine's behavioral effects and that sigma receptor may differentially mediate cocaine-induced hyperactivity and place conditioning.

Sigma-1 receptor ligand PD144418 and sigma-2 receptor ligand YUN-252 attenuate the stimulant effects of methamphetamine in mice.

RATIONALE: Previous research indicates that the selective sigma-1 receptor ligand PD144418 and the selective sigma-2 ligands YUN-252 can inhibit cocaine-induced hyperactivity. The effects of these ligands on other stimulants, such as methamphetamine, have not been reported. OBJECTIVES: The present study examined the effects of PD144418 and YUN-252 pretreatment on methamphetamine-induced hyperactivity after acute treatment. METHODS: Mice (n = 8-14/group) were injected with PD144418 (3.16, 10, or 31.6 µmol/kg), YUN-252 (0.316, 3.16, 31.6 µmol/kg), or saline. After 15 min, mice injected with 2.69 µmol/kg methamphetamine or saline vehicle, where distance traveled during a 60-min period was recorded. Additionally, the effect of PD144418 on the initiation and expression of methamphetamine sensitization was determined by treating mice (n = 8-14/group) with PD144418, methamphetamine or saline repeatedly over a 5-day period, and testing said mice with a challenge dose after a 7-day withdrawal period. RESULTS: Results indicate that both PD144418 and YUN-252, in a dose-dependent manner, attenuated hyperactivity induced by an acute methamphetamine injection. Specifically, 10 µmol/kg or 31.6 µmol/kg of PD144418 and 31 µmol/kg of YUN-252 suppressed methamphetamine-induced hyperactivity. In regard to methamphetamine sensitization, while 10 µmol/kg PD144418 prevented the initiation of methamphetamine sensitization, it did not have an effect on the expression. CONCLUSIONS: Overall, the current results suggest an intriguing potential for this novel sigma receptor ligand as a treatment for the addictive properties of methamphetamine. Future analysis of this novel sigma receptor ligand in assays directly measuring reinforcement properties will be critical.

Analysis and imaging of boron distribution in maize by quantitative neutron capture radiography.

Quantitative neutron capture radiography (QNCR) of 10B found in pre-dried maize samples has been conducted. Calibration standards constructed from filter paper mimicked plant tissues to reduce confounding matrix effects. A mathematical track elimination method improves the LOD as well as the visual contrast image at low boron concentration levels. The LOD for total boron is 1.7 µg/g in a 4 mm2 region of interest (ROI). The w(B) in five individual maize tassel meristems has been determined to be 14.9 µg/g - 21.2 µg/g.

Tactics for preclinical validation of receptor-binding radiotracers.

INTRODUCTION: Aspects of radiopharmaceutical development are illustrated through preclinical studies of [125I]-(E)-1-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA-BF-PE-PIPZE), a radioligand for sigma-1 (σ1) receptors, coupled with examples from the recent literature. Findings are compared to those previously observed for [125I]-(E)-1-(2-(2,3-dimethoxy-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA-DM-PE-PIPZE). METHODS: Syntheses of E-IA-BF-PE-PIPZE and [125I]-E-IA-BF-PE-PIPZE were accomplished by standard methods. In vitro receptor binding studies and autoradiography were performed, and binding potential was predicted. Measurements of lipophilicity and protein binding were obtained. In vivo studies were conducted in mice to evaluate radioligand stability, as well as specific binding to σ1 sites in brain, brain regions and peripheral organs in the presence and absence of potential blockers. RESULTS: E-IA-BF-PE-PIPZE exhibited high affinity and selectivity for σ1 receptors (Ki = 0.43 ± 0.03 nM, σ2/σ1 = 173). [125I]-E-IA-BF-PE-PIPZE was prepared in good yield and purity, with high specific activity. Radioligand binding provided dissociation (koff) and association (kon) rate constants, along with a measured Kd of 0.24 ± 0.01 nM and Bmax of 472 ± 13 fmol/mg protein. The radioligand proved suitable for quantitative autoradiography in vitro using brain sections. Moderate lipophilicity, Log D7.4 2.69 ± 0.28, was determined, and protein binding was 71 ± 0.3%. In vivo, high initial whole brain uptake, >6% injected dose/g, cleared slowly over 24 h. Specific binding represented 75% to 93% of total binding from 15 min to 24 h. Findings were confirmed and extended by regional brain biodistribution. Radiometabolites were not observed in brain (1%). CONCLUSIONS: Substitution of dihydrobenzofuranylethyl for dimethoxyphenethyl increased radioligand affinity for σ1 receptors by 16-fold. While high specific binding to σ1 receptors was observed for both radioligands in vivo, [125I]-E-IA-BF-PE-PIPZE displayed much slower clearance kinetics than [125I]-E-IA-DM-PE-PIPZE. Thus, minor structural modifications of σ1 receptor radioligands lead to major differences in binding properties in vitro and in vivo.

N-phenylpropyl-N'-substituted piperazines occupy sigma receptors and alter methamphetamine-induced hyperactivity in mice.

This study examined the effect of the N-phenylpropyl-N'-substituted piperazine ligands SA4503 (3.4-dimethoxyphenethyl), YZ-067 (4-methoxyphenethyl), YZ-185 (3-methoxyphenethyl) and Nahas-3h (4-methoxybenzyl) on methamphetamine-induced hyperactivity in mice. In a previous study in rats, SA4503 increased methamphetamine-induced hyperactivity at a lower ligand dose and enhanced it at a higher dose. The other ligands have not been investigated in this assay. Presently, mice were administered sigma ligands, and specific [125I]E-IA-DM-PE-PIPZE and [125I]RTI-121 binding was measured to determine σ1 sigma receptor and dopamine transporter occupancy, respectively. Mice were also administered sigma ligands followed by methamphetamine, and locomotor activity was measured. Each of the ligands occupied σ1 sigma receptors (ED50=0.2-0.6µmol/kg) with similar potency, but none occupied the transporter (ED50>10µmol/kg). At the highest dose tested (31.6µmol/kg) all four sigma ligands significantly attenuated methamphetamine-induced hyperactivity. Interestingly, SA4503, YZ-067 and Nahas-3h, but not YZ-185, enhanced methamphetamine-induced hyperactivity at lower ligand doses (1-3.16µmol/kg). These results suggest that these ligands function as stimulant agonists at lower doses and as antagonists at higher does, with subtle changes in the substitution pattern at the 3- and 4-positions of the phenethyl group contributing to the nature of the interactions. Overall, these data indicate a complex role for σ1 sigma receptor ligands in methamphetamine's behavioral effects.

Cocaine occupancy of sigma1 receptors and dopamine transporters in mice.

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 [(125) I]E-N-1-(3'-iodoallyl)-N'-4-(3",4"-dimethoxyphenethyl)-piperazine ([(125) I]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 and 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 [(125) I]3ß-(4-iodophenyl)tropan-2ß-carboxylic acid isopropyl ester ([(125) I]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 [(125) I]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. Nonradioactive 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.

Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503): effects on binding affinity and selectivity for sigma receptors and monoamine transporters.

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 1.75-4.63 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 20-30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13-21 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 (σ1Ki=20.8 nM; σ2Ki=16.4 nM) showed over 100-fold higher DAT affinity (Ki=121 nM) and 6-fold higher SERT affinity (Ki=128nM) 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.

Relationship between cerebral sigma-1 receptor occupancy and attenuation of cocaine's motor stimulatory effects in mice by PD144418.

Psychostimulant effects of cocaine are mediated partly by agonist actions at sigma-1 (σ1) receptors. Selective σ1 receptor antagonists attenuate these effects and provide a potential avenue for pharmacotherapy. However, the selective and high affinity σ1 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 σ1 receptor and dopamine transporter (DAT) occupancy in vivo, and re-examined effects on locomotor activity. PD144418 displayed high affinity for σ1 sites (Ki 0.46 nM) and 3596-fold selectivity over σ2 sites (Ki 1654 nM) in guinea pig brain membranes. No appreciable affinity was noted for serotonin and norepinephrine transporters (Ki >100 µM), and the DAT interaction was weak (Ki 9.0 µM). In vivo, PD144418 bound to central and peripheral σ1 sites in mouse, with an ED50 of 0.22 µmol/kg in whole brain. No DAT occupancy by PD144418 (10.0 µmol/kg) or possible metabolites were observed. At doses that did not affect basal locomotor activity, PD144418 (1, 3.16, and 10 µmol/kg) attenuated cocaine-induced hyperactivity in a dose-dependent manner in mice. There was good correlation (r(2) = 0.88) of hyperactivity reduction with increasing cerebral σ1 receptor occupancy. The behavioral ED50 of 0.79 µmol/kg corresponded to 80% occupancy. Significant σ1 receptor occupancy and the ability to mitigate cocaine's motor stimulatory effects were observed for 16 hours after a single 10.0 µmol/kg dose of PD144418.

Distinctive in vivo kinetics of the new σ1 receptor ligands (R)-(+)- and (S)-(-)-18F-fluspidine in porcine brain.

UNLABELLED: Because of their involvement in growth and survival signaling cascades, the σ(1) receptors (σ(1)Rs) represent a novel target for the treatment of cancer and several brain diseases such as depression and neurodegeneration. From a series of σ1R-specific (18)F-fluoroalkylated spirocyclic piperidines, we have chosen (18)F-fluspidine for detailed investigation of the in vivo kinetics of the (R)-(+)- and (S)-(-)-enantiomers to identify their potential for imaging in humans. METHODS: Enantiopure tosylate precursors for radiolabeling were obtained using chiral preparative high-performance liquid chromatography and used for radiosynthesis of both (18)F-fluspidine enantiomers by nucleophilic substitution with K-(18)F-F-Kryptofix 222-carbonate complex in a synthesis module. Brain pharmacokinetics were investigated by dynamic PET studies in piglets under baseline and blocking conditions using the highly selective σ1R agonist SA4503. Standardized uptake values (SUVs) were calculated for 24 MR-defined brain regions. Total distribution volume (V(T)) and binding potentials (k3'/k4) of (S)-(-)- and (R)-(+)-(18)F-fluspidine were estimated. Furthermore, V(T) values were estimated by graphical analysis using Logan plots. RESULTS: The (S)- and (R)-tosylates were obtained in excellent enantiomeric purities (>98% and >96% enantiomeric excess, respectively). (S)-(-)- and (R)-(+)-(18)F-fluspidine were synthesized within approximately 70 min (radiochemical yield, 35%-45%; specific activity, 650-870 GBq/µmol; radiochemical purity, >99%). Both radiotracers displayed different brain uptake kinetics. Although the initial brain uptake was similar, the SUV at the end of the study differed significantly (P < 0.05), with (R)-(+)-(18)F-fluspidine showing about 60%-150% higher values. Administration of SA4503 reduced SUV almost equally for both radiotracers by approximately 65%. Furthermore, k(3)' was significantly decreased under blocking conditions in almost all regions ((S)-(-)-(18)F-fluspidine, -90%-95%; (R)-(+)-(18)F-fluspidine, -70%-90%) whereas effects on k(4) differed according to the particular brain region. V(T) estimated by both graphical analysis using Logan plots and full nonlinear kinetic analysis revealed significant inhibition for both radiotracers under blocking conditions. CONCLUSION: Both (S)-(-)- and (R)-(+)-(18)F-fluspidine appear to be suitable for σ1R imaging in humans. The different pharmacokinetics of (S)-(-)-(18)F-fluspidine and (R)-(+)-(18)F-fluspidine may have the potential for application in the diagnostics of different pathologic conditions.

A selective sigma-2 receptor ligand antagonizes cocaine-induced hyperlocomotion in mice.

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.

N-Phenylpropyl-N'-(3-methoxyphenethyl)piperazine (YZ-185) Attenuates the Conditioned-Rewarding Properties of Cocaine in Mice.

Sigma receptor antagonists diminish the effects of cocaine in behavioral assays, including conditioned place preference. Previous locomotor activity experiments in mice determined that the sigma receptor ligand YZ-185 (N-phenylpropyl-N'-(3-methoxyphenethyl)piperazine) enhanced cocaine-induced hyperactivity at a lower (0.1 µ mol/kg) dose and dose-dependently attenuated cocaine-induced hyperactivity at higher (3.16-31.6 µ mol/kg) doses. The present study investigated the effect of YZ-185 on cocaine's conditioned-rewarding properties in mice. YZ-185 (0.1, 0.316, 3.16, and 31.6 µ mol/kg) did not have intrinsic activity to produce conditioned place preference or aversion. A higher (31.6 µ mol/kg) YZ-185 dose, but not lower (0.1-3.16 µ mol/kg) YZ-185 doses, prevented the development of place preference to cocaine (66 µ mol/kg). YZ-185 did not alter the expression of cocaine place preference. To further characterize YZ-185's behavioral profile, its effects in the elevated zero maze and rotarod procedures were also determined; YZ-185 produced no significant change from baseline in either assay, indicating that the sigma receptors probed by YZ-185 do not regulate anxiety-like or coordinated motor skill behaviors. Overall, these results suggest that YZ-185 is a sigma receptor antagonist at the 31.6 µ mol/kg dose and demonstrate that sigma receptors can mediate the development of the conditioned-rewarding properties of cocaine.

Effects of N-phenylpropyl-N'-substituted piperazine sigma receptor ligands on cocaine-induced hyperactivity in mice.

The present study examined N-phenylpropyl-N'-substituted piperazine sigma receptor ligands on cocaine-induced changes in locomotor activity in mice. Previous reports indicate that N-phenylpropyl-N'-(4-methoxybenzyl)piperazine (Nahas-3h), N-phenylpropyl-N'-(4-methoxyphenethyl)piperazine (YZ-067), and N-phenylpropyl-N'-(3-methoxyphenethyl)piperazine (YZ-185) bind with high affinity (Ki values≈1 nM) to σ1 sigma receptors. YZ-067 and YZ-185 are known to attenuate cocaine-induced convulsions, while Nahas-3h has not been tested in behavioral studies. Nahas-3h significantly attenuated cocaine-induced hyperactivity. YZ-067 decreased the effect of cocaine in a dose-dependent manner. Interestingly, YZ-185 inhibited cocaine's effect at higher doses, but enhanced cocaine's effect at a low dose. The YZ-185 inhibition of cocaine-induced hyperactivity was not surmounted by increasing the cocaine dose. Overall, this study is consistent with previous work showing the ability of certain sigma receptor ligands to affect cocaine-induced hyperactivity. Further, subtle alterations of ligand structure and the specific dosage levels employed influence the behavioral effects observed, with a 3-methoxy substituent apparently conferring the ability of a ligand to enhance cocaine's locomotor stimulatory effects.

Synthesis, radioiodination and in vitro and in vivo sigma receptor studies of N-1-allyl-N´-4-phenethylpiperazine analogs.

INTRODUCTION: Sigma-1 (σ(1)) receptor radioligands are useful for basic pharmacology studies and for imaging studies in neurology, psychiatry and oncology. We derived a hybrid structure, N-1-allyl-N´-4-phenethylpiperazine, from known ligands TPCNE and SA4503 for use as a scaffold for development of radioiodinated σ(1) receptor ligands. METHODS: E-and Z-N-1-(3'-iodoallyl)-N´-4-(3″,4″-dimethoxyphenethyl)-piperazine (E-1 and Z-1), N-1-allyl-N´-4-(3',4'-dimethoxyphenethyl)-piperazine (2) and E-N-1-(3'-iodoallyl)-N´-4-(3″-methoxy-4'´-hydroxyphenethyl)-piperazine (3) were synthesized. Affinities for σ(1) and σ(2) receptors were determined. [(125)I]E-1 and [(125)I]Z-1 were prepared and evaluated in vivo in mice. [(125)I]E-1 was further evaluated in σ(1) receptor binding assays in vitro. RESULTS: E-1 displayed moderately high apparent affinity (15 nM) for σ(1) sites and 84-fold selectivity against σ(2) sites. Z-1 showed similar σ(1) affinity, but only 23-fold selectivity. In contrast, 2 exhibited poor binding to both subtypes, while 3 had good affinities but poor selectivity. E-1 profiled as a probable antagonist in the phenytoin shift assay. [(125)I]E-1 and [(125)I]Z-1 were prepared in good yields and with high specific radioactivities. Log D(7.4) values (2.25 and 2.27) fall within the optimal range for in vivo studies. Both radioligands selectively labeled σ(1) receptors in mouse brain and peripheral organs in vivo. [(125)I]E-1 showed a higher level of specific binding than [(125)I]Z-1 and displayed good metabolic stability. Further, [(125)I]E-1 selectively labeled σ(1) receptors in mouse brain homogenates (K(d) 3.79 nM; B(max)=599 fmol/mg protein). CONCLUSIONS: [(125)I]E-1 is a selective σ(1) receptor radioligand that exhibits properties amenable to in vitro and in vivo studies, with possible extension to single photon emission computed tomography using iodine-123.

Effect of structural modification in the amine portion of substituted aminobutyl-benzamides as ligands for binding σ1 and σ2 receptors.

5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide (1) is one of the most potent and selective σ(2) receptor ligands reported to date. A series of new analogs, where the amine ring fused to the aromatic ring was varied in size (5-7) and the location of the nitrogen in this ring was modified, has been synthesized and assessed for their σ(1)/σ(2) binding affinity and selectivity. The binding affinity of an open-chained variant of 1 was also evaluated. Only the five-membered ring congener of 1 displayed a higher σ(1)/σ(2) selectivity, derived from a higher σ(2) affinity and a lower σ(1) affinity. Positioning the nitrogen adjacent to the aromatic ring in the five-membered and six-membered ring congeners dramatically decreased affinity for both subtypes. Thus, location of the nitrogen within a constrained ring is confirmed to be key to the exceptional σ(2) receptor binding affinity and selectivity for this active series.

The sigma receptor agonist SA4503 both attenuates and enhances the effects of methamphetamine.

BACKGROUND: Methamphetamine's behavioral effects have been attributed to its interaction with monoamine transporters; however, methamphetamine also has affinity for sigma receptors. METHOD: The present study investigated the effect of the sigma receptor agonist SA 4503 and the sigma receptor antagonists BD-1047 and BD-1063 on methamphetamine-evoked [(3)H]dopamine release from preloaded rat striatal slices. The effect of SA 4503 on methamphetamine-induced hyperactivity and on the discriminative stimulus properties of methamphetamine also was determined. RESULTS: SA 4503 attenuated methamphetamine-evoked [(3)H]dopamine release in a concentration-dependent manner. BD-1047 and BD-1063 did not affect release. SA 4503 dose-dependently potentiated and attenuated methamphetamine-induced hyperactivity. SA 4503 pretreatment augmented the stimulus properties of methamphetamine. CONCLUSIONS: Our findings indicate that SA 4503 both enhances and inhibits methamphetamine's effects and that sigma receptors are involved in the neurochemical, locomotor stimulatory and discriminative stimulus properties of methamphetamine.

SA 4503 attenuates cocaine-induced hyperactivity and enhances methamphetamine substitution for a cocaine discriminative stimulus.

Cocaine exhibits preferential (~15-fold) affinity for σ1 over σ2 sigma receptors, and previous research has shown an interaction of σ1 receptor-selective ligands and cocaine's behavioral effects. The present study investigated the effect of the putative sigma receptor agonist SA 4503 (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride) on cocaine's locomotor stimulatory and discriminative stimulus properties. At doses without intrinsic activity, SA 4503 dose-dependently attenuated cocaine-induced hyperactivity in mice. This inhibition was overcome by increasing the cocaine dose. In rats trained to use cocaine as a discriminative stimulus in a drug discrimination task, doses of SA 4503 that did not substitute for the cocaine stimulus did not alter the cocaine substitution curve. However, SA 4503 potentiated the effect of methamphetamine to substitute for the cocaine stimulus. These data support a role for sigma receptors in the locomotor-activating properties of cocaine and, importantly, indicate a role for these receptors in the discriminative stimulus effects of methamphetamine. The data also suggest sigma receptors mediate the activity of different dopamine pathways responsible for the behavioral effects of psychostimulants.

Synthesis and σ1 Receptor Binding of Halogenated N,N'-Diphenethylethylenediamines.

Eight halogenated N,N'-diphenethylethylenediamines were synthesized, characterized and evaluated for σ1 receptor binding affinity in vitro. Measurements of lipophilicity also were obtained. The substitution pattern on one of the aromatic rings remained constant as 3,4-dichloro, while the substituents on the other aromatic ring were varied to include fluorine, bromine or iodine in either the 2-, 3- or 4- positions. Two main structure activity relationships were observed. First, halogen substitution on the 3- or 4-positions of the aromatic ring conferred higher binding affinities (Ki values 6.35 - 15.82 nM) than the corresponding substitutions at the 2-position (Ki values 12.08 - 43.15 nM). Second, derivatives containing either a bromo or fluoro substituent at a given position showed higher σ1 receptor binding affinities than derivatives with a corresponding iodo substituent. The data indicate that σ1 receptor affinity for this structural series is sensitive to steric bulk at the 2-position. Log k'w measurements for the halogenated N,N'-diphenethylethylenediamines were determined by high performance liquid chromatography, and varied from 2.54 - 3.71. In particular, the 3-fluoro analog exhibited a log k'w = 2.54 accompanied by a σ1 receptor Ki = 7.8 nM. These novel N,N'-diphenethylethylenediamines warrant further investigation in behavioral assays, and radiolabeled versions may prove suitable for in vivo studies of σ1 receptors.

Design, synthesis, and biological evaluation of an antagonist-bombesin analogue as targeting vector.

The gastrin releasing peptide receptor (GRP-R) is overexpressed on a number of tumors and cancer cell lines including pancreas, prostate, breast, gastrointestinal, and small cell lung cancer (SCLC). Radiolabeled bombesin (BBN) analogues have exhibited high binding affinity and specificity to the GRP-R. A bombesin analogue with an antagonist targeting vector at the C-terminus, DOTA-aminohexanoyl-[D-Phe(6), Leu-NHCH 2CH 2CH3(13), des Met(14)] BBN[6-14] (1, "Bomproamide"), has been synthesized and displays high binding affinity (IC50 = 1.36 +/- 0.09 nM) against (125)I-Tyr (4)-BBN in in vitro competitive assays using PC-3 cells. Maximum internalization of (111)In-1 reached 14% in PC-3 cells after 45 min of incubation. Rapid (0.25 h PI) and high (12.21 +/- 3.2%ID/g) pancreatic uptake of (111)In-1 was observed in healthy CF-1 mice, and 90% of the activity was blocked by coinjection of 100 mug of BBN. Rapid (0.25 h PI) and high uptake (6.90 +/- 1.06%ID/g) was observed in PC-3 prostate cancer xenografts in SCID mice, as well as visualized clearly in a SPECT/CT study. These results support the use of a bombesin construct with an antagonist C-terminal vector as a candidate of choice for specific in vivo imaging of tumors overexpressing GRP-receptors.

Molecular imaging of bcl-2 expression in small lymphocytic lymphoma using 111In-labeled PNA-peptide conjugates.

UNLABELLED: The bcl-2 gene is overexpressed in non-Hodgkin's lymphoma (NHL), such as small lymphocytic lymphoma (SLL), and many other cancers. Noninvasive imaging of bcl-2 expression has the potential to identify patients at risk for relapse or treatment failure. The purpose of this study was to synthesize and evaluate radiolabeled peptide nucleic acid (PNA)-peptide conjugates targeting bcl-2 gene expression. An (111)In-labeled PNA complementary to the translational start site of bcl-2 messenger RNA was attached to Tyr(3)-octreotate for somatostatin receptor-mediated intracellular delivery. METHODS: DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate (1) and 3 control conjugates (DOTA-nonsense-PNA-Tyr(3)-octreotate (2), DOTA-anti-bcl-2-PNA-Ala[3,4,5,6]-substituted congener (3), and DOTA-Tyr(3)-octreotate (4) [DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid]) were synthesized by standard solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. In vitro studies were performed in Mec-1 SLL cells, which express both bcl-2 messenger RNA and somatostatin receptors. Biodistributions and microSPECT/CT studies were performed in Mec-1-bearing SCID (severe combined immunodeficiency) mice, a new animal model of human SLL. RESULTS: (111)In-Labeled conjugate 1 was taken up by Mec-1 cells through a somatostatin receptor-mediated mechanism. Biodistribution studies showed specific tumor uptake of conjugate 1, the somatostatin analog 4, and the PNA nonsense conjugate 2, but not of the mutant peptide conjugate 3. Mec-1 tumors could be detected by microSPECT/CT using (111)In-labeled DOTA-Tyr(3)-octreotate (4) and the targeted anti-bcl-2 conjugate (1), but not using the 2 negative control conjugates 2 and 3. CONCLUSION: A new (111)In-labeled antisense PNA-peptide conjugate demonstrated proof of principle for molecular imaging of bcl-2 expression in a new mouse model of human SLL. This imaging agent may be useful for identifying NHL patients at risk for relapse and conventional treatment failure.

Synthesis and structure-activity relationships of N-(3-phenylpropyl)-N'-benzylpiperazines: Potent ligands for sigma1 and sigma2 receptors.

Ten N-(3-phenylpropyl)-N'-benzylpiperazines having different substituents on the benzyl moiety were synthesized and evaluated for sigma(1) and sigma(2) receptor binding. The sigma(1) affinities were 0.37-2.80nM, sigma(2) affinities were 1.03-34.3nM, and selectivities, as sigma(2)/sigma(1) affinity ratios, ranged from 1.4 to 52. Three compounds tested in a phenytoin shift binding assay profiled as probable sigma(1) antagonists. Quantitative structure-activity relationships depended on pi(x), MR or E(s) and Hammett sigma values. The hydrophobicity term is negative for sigma(1) binding but positive for sigma(2) binding, indicating a major difference between the pharmacophores.