Sigma-2 receptor ligand as a novel method for delivering a SMAC mimetic drug for treating ovarian cancer.

BACKGROUND: The sigma-2 receptor has been validated as a biomarker for proliferating tumours. Second mitochondria-derived activator of caspase (Smac) is a protein released from mitochondria into the cytosol, leading to apoptosis. In this study, we investigated a sigma-2 ligand as a tumour-targeting drug delivery agent for treating ovarian cancer. METHODS: A sigma-2 ligand, SW 43, was conjugated with a Smac mimetic compound (SMC), SW IV-52s, to form SW III-123. The delivery function of the sigma-2 moiety and cell killing mechanisms of SW III-123 were examined in human ovarian cancer cell lines. RESULTS: SW III-123 internalisation into ovarian cancer cells was mediated by sigma-2 receptors. SW III-123, but not SW IV-52s or SW 43, exhibited potent cytotoxicity in human ovarian cancer cell lines SKOV-3, CaOV-3 and BG-1 after 24-h treatment, suggesting that the sigma-2 ligand successfully delivered SMC into ovarian cancer cells. SW III-123 induced rapid degradation of inhibitor of apoptosis proteins (cIAP1 and cIAP2), accumulation of NF-κB-inducing kinase (NIK) and phosphorylation of NF-κB p65, suggesting that SW III-123 activated both canonical and noncanonical NF-κB pathways in SKOV-3 cells. SW III-123 cleaved caspase-8, -9 and -3. Tumour necrosis factor alpha (TNFα) antibody markedly blocked SW III-123-induced cell death and caspase-3 activity in SKOV-3 cells, indicating that SW III-123 activated both intrinsic and extrinsic apoptotic pathways and induced TNFα-dependent cell death in SKOV-3 cells. CONCLUSION: Sigma-2 ligands are a promising tumour-targeting drug delivery agent. Sigma-2-conjugated SMC exemplifies a novel class of therapeutic drugs for treating ovarian cancer.

Sigma-2 ligands induce tumour cell death by multiple signalling pathways.

BACKGROUND: The sigma-2 receptor has been identified as a biomarker of proliferating cells in solid tumours. In the present study, we studied the mechanisms of sigma-2 ligand-induced cell death in the mouse breast cancer cell line EMT-6 and the human melanoma cell line MDA-MB-435. METHODS: EMT-6 and MDA-MB-435 cells were treated with sigma-2 ligands. The modulation of multiple signaling pathways of cell death was evaluated. RESULTS: Three sigma-2 ligands (WC-26, SV119 and RHM-138) induced DNA fragmentation, caspase-3 activation and PARP-1 cleavage. The caspase inhibitor Z-VAD-FMK partially blocked DNA fragmentation and cytotoxicity caused by these compounds. These data suggest that sigma-2 ligand-induced apoptosis and caspase activation are partially responsible for the cell death. WC-26 and siramesine induced formation of vacuoles in the cells. WC-26, SV119, RHM-138 and siramesine increased the synthesis and processing of microtubule-associated protein light chain 3, an autophagosome marker, and decreased the expression levels of the downstream effectors of mammalian target of rapamycin (mTOR), p70S6K and 4EBP1, suggesting that sigma-2 ligands induce autophagy, probably by inhibition of the mTOR pathway. All four sigma-2 ligands decreased the expression of cyclin D1 in a time-dependent manner. In addition, WC-26 and SV119 mainly decreased cyclin B1, E2 and phosphorylation of retinoblastoma protein (pRb); RHM-138 mainly decreased cyclin E2; and 10 µM siramesine mainly decreased cyclin B1 and pRb. These data suggest that sigma-2 ligands also impair cell-cycle progression in multiple phases of the cell cycle. CONCLUSION: Sigma-2 ligands induce cell death by multiple signalling pathways.

Sigma 2 receptors as potential biomarkers of proliferation in breast cancer.

sigma 1 and sigma 2 receptors have been shown to exist in a number of rodent and human tumor cell lines. Although their expression is heterogeneous and their function is unknown, sigma receptors have been proposed as potential targets for diagnostic tumor-imaging agents. In this study, the density of sigma 2 receptors in proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66, was examined. Scatchard analyses of sigma 2 receptors were performed on membrane preparations of 66 P cells from 3-day cultures and 66 Q cells from 7-, 10-, and 12-day cultures. The Scatchard studies revealed that 66 P cells had approximately 10 times more sigma 2 receptors/cell than the 66 Q cells from 10-day cultures. Although > 97% of the cells were quiescent after 7 days in culture, the maximum differential in the sigma 2 expression between 66 P and 66 Q cells was not attained until these cells had been in culture for 10 days. These data suggest that ligands labeled with positron-emitting or single photon-emitting radionuclides, which selectively bind sigma 2 receptors, have the potential to noninvasively assess the proliferative status of human breast tumors.

Antibodies with high affinity for spiroperidol--II. Cross reactivity with iodobenzamide and domperidone.

Three radioligands, 3H-spiroperidol (3H-SPD), 3H-domperidone (3H-DOMP) and 125I-iodobenzamide (125I-IBZM), were used to investigate the antibody response to two haptens, aminospiroperidol (NH2SPD) and N-aminophenethylspiroperidol (NAPS). Although structurally different, these three radioligands each bind with high affinity to the D2 dopamine receptor. Antibodies with high affinity for 3H-SPD were elicited in rabbits following immunization with the hapten NH2SPD covalently linked to keyhole limpet hemocyanin (KLH). In addition, antibodies in the rabbit anti-NH2SPD antisera bound 125I-IBZM or 3H-DOMP. Rabbit anti-NH2SPD antibodies that bound 125I-IBZM or 3H-DOMP were found to have higher affinity for IBZM or DOMP, respectively, than for SPD. The binding properties of the anti-NH2SPD antibodies that bound 3H-SPD, 125I-IBZM and 3H-DOMP were characterized using a panel of competitive inhibitors and each radioligand appeared to bind to a distinct subpopulation of anti-NH2SPD antibodies. BALB/c mice were immunized with NH2SPD-KLH or NAPS-KLH. A population of antibodies that bound 3H-SPD and a population of antibodies that bound 3H-DOMP were detected. The population of antibodies that bound 3H-DOMP was found to be heteroclitic for DOMP, since DOMP was a more effective competitive inhibitor than SPD. Binding sites for 125I-IBZM were not detected in either the anti-NH2SPD or the anti-NAPS BALB/c antisera. However, two anti-NAPS monoclonal antibodies, N6-24 and N6-29, that bind 3H-SPD with high affinity (Kd = 10(-9) M), were also found to bind IBZM (Ki = 2 x 10(-7) M) and DOMP (Ki = 2 x 10(-6) M). Although anti-NH2SPD and anti-NAPS antibodies were identified that appeared to bind 3H-SPD, 3H-DOMP or 125I-IBZM with high affinity, none of the populations of polyclonal antibodies or monoclonal antibodies bound all three ligands with high affinity.

Bitropic D3 Dopamine Receptor Selective Compounds as Potential Antipsychotics.

Neuropsychiatric disorders represent a substantial social and health care issue. The National Institutes of Health estimates that greater than 2 million adults suffer from neuropsychiatric disorders in the USA. These individuals experience symptoms that can include auditory hallucinations, delusions, unrealistic beliefs and cognitive dysfunction. Although antipsychotic medications are available, suboptimal therapeutic responses are observed for approximately one-third of patients. Therefore, there is still a need to explore new pharmacotherapeutic strategies for the treatment of neuropsychiatric disorders. Many of the medications that are used clinically to treat neuropsychiatric disorders have a pharmacological profile that includes being an antagonist at D2-like (D2, D3 and D4) dopamine receptor subtypes. However, dopamine receptor subtypes are involved in a variety of neuronal circuits that include movement coordination, cognition, emotion, affect, memory and the regulation of prolactin. Consequently, antagonism at D2-like receptors can also contribute to some of the adverse side effects associated with the long-term use of antipsychotics including the a) adverse extrapyramidal symptoms associated with the use of typical antipsychotics and b) metabolic side effects (weight gain, hyperglycemia, increased risk of diabetes mellitus, dyslipidemia and gynecomastia) associated with atypical antipsychotic use. Preclinical studies suggest that D3 versus D2 dopamine receptor selective compounds might represent an alternative strategy for the treatment of the symptoms of schizophrenia. In this review we discuss a) how bitropic Nphenylpiperazine D3 dopamine receptor selective compounds have been developed by modification of the primary (orthosteric) and secondary (allosteric or modulatory) pharmacophores to optimize D3 receptor affinity and D2/D3 binding selectivity ratios and b) the functional selectivity of these compounds. Examples of how these compounds might be modified to develop bivalent ligands capable of interacting with receptor dimers or oligomers are also provided. Preclinical studies using bitropic D3 dopamine receptor selective ligands are also discussed as strategy to pharmacologically dissect the role of the D2 and D3 dopamine receptor subtypes in animal models of neuropsychiatric, neurological and substance abuse disorders. This research has the potential to a) advance the understanding of the role of the D2 and D3 dopamine receptor subtypes in neuropsychiatric disorders and b) lead to new treatment strategies for neuropsychiatric disorders.

Morphine-induced spinal cholinergic activation: in vivo imaging with positron emission tomography.

Positron emission tomography (PET) imaging of spinal cord in monkeys with a cholinergic tracer demonstrates increased spinal cholinergic activity in response to an analgesic dose of morphine, and this PET result correlates with measurement of acetylcholine spillover into spinal cord extracellular space induced by morphine, as measured by microdialysis. Previous studies in rats, mice, and sheep demonstrate activation of spinal cholinergic neurons by systemic opioid administration, and participation of this cholinergic activity in opioid-induced analgesia. Testing the relevance of this observation in humans has been limited to measurement of acetylcholine spillover into lumbar cerebrospinal fluid. The purpose of this study was to apply a recently developed method to image spinal cholinergic terminals non-invasively via PET and to test the hypothesis that the tracer utilized would reflect changes in local cholinergic activity. Following Animal Care and Use Committee approval, seven adult male rhesus monkeys were anesthetized on three separate occasions. On two of the occasions PET scans were performed using [(18)F] (+)-4-fluorobenzyltrozamicol ([(18)F]FBT), which selectively binds to the vesicular acetylcholine (ACh) transporter in the presynaptic cholinergic terminals. PET scans were preceded by injection of either saline or an analgesic dose of IV morphine (10 mg/kg). On the third occasion, microdialysis catheters were inserted in the spinal cord dorsal horn and acetylcholine concentrations in dialysates determined before and after IV morphine injection. Morphine increased cholinergic activity in the spinal cord, as determined by blood flow corrected distribution volume of [(18)F]FBT in the cervical cord compared to the cerebellum. Morphine also increased acetylcholine concentrations in microdialysates from the cervical cord dorsal horn. The one animal which did not show increased spinal cholinergic activity by PET from this dose of morphine also did not show increased acetylcholine from this morphine dose in the microdialysis experiment. These data confirm the ability to use PET to image spinal cholinergic terminals in the monkey spinal cord and suggest that acute changes in cholinergic activity can be imaged with this non-invasive technique. Following preclinical screening, PET scanning with [(18)F]FBT may be useful to investigate mechanisms of analgesic action in normal humans and in those with pain.

PET imaging of dopamine D2 receptors with [18F]fluoroclebopride in monkeys: effects of isoflurane- and ketamine-induced anesthesia.

The purpose of the present study was to determine whether positron emission tomography (PET) studies in monkeys with the dopamine (DA) D2 receptor ligand [18F]fluoroclebopride (FCP) would be significantly influenced by anesthetic induction with isoflurane (approximately 5.0%) compared to induction with 10 mg/kg ketamine. Five experimentally-naive adult male cynomolgus monkeys (Macaca fascicularis) were trained to sit calmly in a primate restraint chair. Before the first PET scan, each monkey was anesthetized, by mask, with isoflurane. After complete sedation, the monkey was intubated and anesthesia was maintained throughout the PET study by isoflurane (approximately 1.5%). At least 1 month later, a second PET study was conducted in which anesthesia was induced with ketamine and maintained by isoflurane (approximately 1.5%). Irrespective of induction anesthetic, there was a high uptake of [18F]FCP and a linear rate of washout from the basal ganglia for all monkeys. There were also no differences in time to peak uptake (approximately 25 min), in clearance half-life (t1/2 = 140-164 min) or in D2 binding (distribution volume ratios of 2.48 vs. 2.50). These results indicate that induction anesthetic did not differentially affect D2 binding of [18F]FCP in monkeys. Furthermore, the low variability between studies indicates that [18F]FCP is an excellent ligand for longitudinal studies of D2 receptors in nonhuman primates.

Synthesis, chemical reactivity, and antileukemic activity of 5-substituted 6,7-bis(hydroxymethyl)pyrrolo[1,2-c]thiazole biscarbamates and the corresponding sulfoxides and sulfones.

A series of bis(N-methylcarbamate) and bis[N-(2-propyl)carbamate] derivatives of 5-substituted 6,7-bis(hydroxy-methyl)pyrrolo[1,2-c]thiazoles was prepared. The compounds were tested for activity in vivo against P388 lymphocytic leukemia, and the chemical reactivities of the compounds were compared by using the model nucleophile 4-(4-nitrobenzyl)pyridine (NBP). The 5-(3,4-dichlorophenyl)-substituted biscarbamates 6b, 8b, and 12b were inactive and unreactive toward NBP. The 5-methyl-substituted biscarbamates 6a, 7a, 8a, 9a, 12a, and 13a were all active against murine P388 lymphocytic leukemia. The chemical reactivities of the active compounds depended on the oxidation state of the sulfur. The reactivity toward NBP followed the order S greater than SO much greater than SO2. The sulfones 12a and 13a are the most active compounds in this series, and their lack of reactivity toward NBP led to the suggestion that 12a and 13a are activated in vivo.

Synthesis and structure-activity relationships of N-(1-benzylpiperidin-4-yl)arylacetamide analogues as potent sigma1 receptor ligands.

A series of N-(1-benzylpiperidin-4-yl)arylacetamides were synthesized and evaluated for their binding properties for sigma1 and sigma2 receptors. In agreement with previously reported sigma1/sigma2 receptor binding data for N-(1-benzylpiperidin-4-yl)phenylacetamide, all of the N-(1-benzylpiperidin-4-yl)arylacetamide compounds reported below displayed higher affinity for sigma1 vs sigma2 receptors. Replacement of the phenyl ring of the phenylacetamide moiety with a thiophene, naphthyl, or indole aromatic ring had no significant effect on the sigma1 receptor affinity. Replacement of the phenyl ring with an imidazole or pyridyl aromatic ring resulted in a >60-fold loss in affinity for sigma1 receptors and no significant binding affinity for sigma2 receptors. Substitution on the aromatic ring of the benzyl group showed a similar or slightly decreased affinity for sigma1 receptors. Substitution on the aromatic rings of both the phenylacetamide moiety and the benzyl group with a halogen resulted in a similar affinity for sigma(1) receptors and a significantly increased affinity for sigma2 receptors. Comparative molecular field analysis revealed that electrostatic properties of the substituents in the phenylacetamide aromatic ring strongly influenced binding to sigma1 receptors. Compounds 1, 10, 18, 22, 37, and 40 showed the highest selectivity for sigma1 receptors with K(i) (sigma2) to K(i) (sigma(1)) ratios of 100, >92, >122, 77, 74, and 80, respectively. In agreement with previously reported results, the phenylacetamide analogues had no binding affinity for dopamine receptors (D2/D3).

Synthesis and structure-activity relationships of naphthamides as dopamine D3 receptor ligands.

A series of naphthamides were synthesized, and the affinities of these compounds were determined for dopamine D2 and D3 receptors using radioligand binding techniques. The naphthamide compounds that were prepared include N-(1-alkylpiperidin-4-yl)-4-bromo-1-methoxy-2-naphthamides (1-6), (S)-N-(1-alkylpyrrolidin-3-yl)-4-bromo-1-methoxy-2-naphthamides (7-12), (R)-N-(1-alkylpyrrolidin-3-yl)-4-bromo-1-methoxy-2-naphthamides (13-18), (S)-N-(1-alkyl-2-pyrrolidinylmethyl)-4-bromo-1-methoxy-2-naphthamides (19-25), (R)-N-(1-alkyl-2-pyrrolidinylmethyl)-4-bromo-1-methoxy-2-naphthamides (26-31), and N-(9-alkyl-9-azabicyclo[3.3.1]nonan-3beta-yl)-4-bromo-1-methoxy-2-naphthamides (32, 33). The results of in vitro radioligand binding studies indicated that the majority of the naphthamide analogues bound with high affinity at both the D2 and D3 dopamine receptor subtypes and most of the compounds demonstrated some selectivity for the dopamine D3 dopamine receptor subtype. These results demonstrated that both the structure of the central amine moiety (piperidine, pyrrolidine, and 9-azabicyclo[3.3.1]nonane) ring and the N-(alkyl) substitution on the amine significantly effects the binding affinity at D2 and D3 dopamine receptors. The bulkiness of the N-(1-alkyl) substituent was found to (a) have no effect on pharmacologic selectivity, (b) increase the affinity at D3 receptors, or (c) decrease the affinity at D2 receptors. The most potent analogue in this series was (S)-N-(1-cycloheptylpyrrolidin-3-yl)-4-bromo-1-methoxy-2-naphthamide (10), which had equilibrium dissociation (K(i)) values of 1.8 and 0.2 nM for D2 and D3 receptors, respectively. The most selective analogue was (R)-N-(1-cycloheptyl-2-pyrrolidinylmethyl)-4-bromo-1-methoxy-2-naphthamide (30), which had K(i) values of 62.8 and 2.4 nM for D2 and D3 receptors, respectively. Radioligand binding results for sigma receptors indicated that the structure of the amine moiety and the N-(1-alkyl) substitutions also significantly influence the affinity and selectivity of these compounds at the sigma1 and sigma2 sigma receptor subtypes. The two naphthamides containing a 9-azabicyclo[3.3.1]nonan-3beta-yl central ring were found to be selective for sigma2 receptors.

Hydroxylated decahydroquinolines as ligands for the vesicular acetylcholine transporter: synthesis and biological evaluation.

Analogues of the potent anticholinergic 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1) in which the cyclohexyl fragment was replaced with an N-acyl or N-alkyl trans-decahydroquinolyl moiety were synthesized and evaluated as potential ligands for the vesicular acetylcholine transporter (VAChT). The binding of compounds, such as 18, 20, and 21, was both stereospecific and of comparable magnitude to that of the closely related vesamicol analogue 2,3-trans-4a, 8a-trans-3-hydroxy-2-(4-phenylpiperidino)-1,2,3,4,5,6,7, 8-decahydronaphthalene (6) which displays subnanomolar affinity for this transporter. However, these compounds also demonstrated high affinities for sigma(1) and sigma(2) receptors and thus failed to show significantly improved selectivity over previously reported vesamicol analogues.

Dopamine D-2 receptor imaging radiopharmaceuticals: synthesis, radiolabeling, and in vitro binding of (R)-(+)- and (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N- [(1-ethyl-2-pyrrolidinyl)methyl]benzamide.

In developing central nervous system (CNS) dopamine D-2 receptor imaging agents, enantiomers, R-(+) and S-(-) isomers, of 3-[125I]iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2- pyrrolidinyl)methyl]benzamide, [125I]IBZM, were synthesized, and their in vitro binding characteristics were evaluated in rat striatum tissue preparation. The (S)-(-)-[125I]IBZM showed high specific dopamine D-2 receptor binding (Kd = 0.43 nM, Bmax = 0.48 pmol/mg of protein). Competition data of various ligands for IBZM binding displayed the following rank order of potency: spiperone greater than (S)-(-)-IBZM greater than (+)-butaclamol much greater than (R)-(+)-IBZM greater than (S)-(-)-BZM greater than dopamine greater than ketanserin greater than SCH23390 much greater than propanolol. The results indicate that [125I]IBZM binds specifically to the dopamine D-2-receptor with stereospecificity. The [123I]IBZM is potentially useful as an imaging agent for the investigation of dopamine D-2 receptors in humans.

Effect of N-alkylation on the affinities of analogues of spiperone for dopamine D2 and serotonin 5-HT2 receptors.

Two series of N-substituted spiperone analogues were prepared and evaluated in vitro to measure their affinities for dopamine D2 and serotonin 5-HT2 receptors. Substitution of the amide nitrogen with an alkyl group of five carbon units or less resulted in analogues displaying a low selectivity for D2 compared to 5-HT2 receptors. However, a moderate improvement in selectivity for D2 receptors was observed with N-benzylspiperone. Substitution at either the ortho or para position of the benzyl group resulted in a further reduction in affinity for 5-HT2 receptors and improvement in the selectivity ratio. Examination of N-substituted analogues of spiperone may provide insights into the topography of the antagonist binding region of the 5-HT2 receptor. The results also suggest that an 18F-labeled analogue of N-(4-nitrobenzyl)spiperone (4p) may be a suitable tracer for studying D2 receptors with positron emission tomography since this compound displays a high selectivity for D2 receptors relative to that of spiperone and N-methylspiperone.

N-hydroxyalkyl derivatives of 3 beta-phenyltropane and 1-methylspiro[1H-indoline-3,4'-piperidine]: vesamicol analogues with affinity for monoamine transporters.

As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter ligand 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives of 3 beta-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4'-piperidine], 7, were synthesized and tested for binding in vitro. Although a few compounds displayed moderately high affinity for the vesicular acetylcholine transporter, no compound was more potent than the prototypical vesicular acetylcholine transporter ligand vesamicol. However, a few derivatives of 6 displayed higher affinity for the dopamine transporter than cocaine. We conclude that modification of the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter ligands.

Synthesis and quantitative structure-activity relationships of N-(1-benzylpiperidin-4-yl)phenylacetamides and related analogues as potent and selective sigma1 receptor ligands.

A series of N-(1-benzylpiperidin-4-yl)phenylacetamide derivatives was synthesized and evaluated for affinity at sigma1 and sigma2 receptors. Most of these compounds showed a high affinity for sigma1 receptors and a low to moderate affinity for sigma2 receptors. The unsubstituted compound N-(1-benzylpiperidin-4-yl)phenylacetamide, 1, displayed a high affinity and selectivity for sigma1 receptors (Ki values of 3.90 nM for sigma1 receptors and 240 nM for sigma2 receptors). The influence of substitutions on the phenylacetamide aromatic ring on binding at both the sigma1 and sigma2 receptor has been examined through Hansch-type quantitative structure-activity relationship (QSAR) studies. In general, all 3-substituted compounds, except for the OH group, had a higher affinity for both sigma1 and sigma2 receptors when compared with the corresponding 2- and 4-substituted analogues. The selectivity for sigma1 receptors displayed a trend of 3 > 2 approximately 4 for Cl, Br, F, NO2, and OMe substituted analogues. Halogen substitution on the aromatic ring generally increased the affinity for sigma2 receptors while maintaining a similar affinity for sigma1 receptors. Substitution with electron-donating groups, such as OH, OMe, or NH2, resulted in weak or negligible affinity for sigma2 receptors and a moderate affinity for sigma1 receptors. The 2-fluoro-substituted analogue, 11, exhibited the highest selectivity for sigma1 receptors among all compounds tested, with a Ki value of 3.56 nM for sigma1 receptors and 667 nM for sigma2 receptors. Compounds 1, 5, 9, 11, and 20 had no affinity for dopamine D2 (IC50 > 10 000 nM) and D3 (IC50 > 10 000 nM) receptors. The nanomolar binding affinity and high selectivity for sigma1 receptors suggest that these compounds may be developed as potential radiotracers for positron emission tomography or single photon emission computerized tomography imaging studies.

18F-labeled benzamides for studying the dopamine D2 receptor with positron emission tomography.

Two series of (N-benzylpiperidin-4-yl)- and (9-azabicyclo[3.3.1]nonan- 3 beta-yl)benzamides were prepared, and in vitro binding assays were used to measure the affinity of these compounds for dopamine D2, dopamine D3, serotonin 5-HT2, and alpha 2-adrenergic receptors. The results of these studies indicated compounds 23, 26b, and 34 have the selectivity needed for in vivo studies of the D2 (and possibly D3) receptors. 18F-Labeled analogues of 23, 26b and 34 were prepared by N-alkylation of the corresponding desbenzyl precursors with [18F]-4-fluorobenzyl iodide. Preliminary in vivo studies demonstrated that [18F]-23 and [18F]-26b are suitable candidates for further evaluation in positron emission tomography imaging studies. The slow rate of washout of [18F]-34 from nondopaminergic regions and its comparatively high lipophilicity indicates that this compound may not be suitable for imaging studies because of a high level of nonspecific binding.

Reproducibility of repeated measures of cholinergic terminal density using.

UNLABELLED: [18F](+)-4-fluorobenzyltrozamicol (FBT), which selectively binds to the vesicular acetylcholine transporter in the presynaptic cholinergic neuron, has previously been shown to be a useful ligand for the study of cholinergic terminal density in the basal ganglia with PET. The goal of this study was to assess the test-retest variability of [18F]FBT and PET measurements under baseline conditions in the basal ganglia. METHODS: After approval from the Animal Care and Use Committee, 6 rhesus monkeys underwent a series of 2 [18F]FBT PET scans (time between scans, 32-301 d) under isoflurane anesthesia. Each scan was initiated on the bolus injection of the radiotracer and consisted of 26 frames acquired during 180 min. Arterial blood samples were collected over the course of each scan to determine the metabolite-corrected arterial input function. Tissue time-activity curves were obtained from the scan data by drawing regions of interest over the basal ganglia and cerebellum. The distribution volume ratio for the basal ganglia was then determined for each scan by taking the ratio of the basal ganglia (specific binding) to cerebellum (nonspecific binding) distribution volume. Distribution volumes were derived using the Logan graphic analysis technique as well as a standard 3-compartment model. Additionally, the radioactivity concentration ratio was calculated as the ratio of the average [18F]FBT concentration in the basal ganglia to that in the cerebellum during the last half of the study (85-170 min). The constant K1, determined using the standard 3-compartment model, was used as an index of blood flow changes between studies. RESULTS: For all subjects, the test-retest variability was less than 15% for the distribution volume ratio and 12% for the radioactivity concentration ratio. Good agreement was found between the distribution volume ratio calculated using the graphic technique and the standard 3-compartment model. Using K1 as an index, the variability in blood flow seen in both the basal ganglia and the cerebellum was significantly reduced in their ratio. CONCLUSION: These results show the reproducibility of [18F]FBT and PET measurements in the basal ganglia.

Vesamicol analogues as sigma ligands. Molecular determinants of selectivity at the vesamicol receptor.

The present study compares the affinities of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1) and selected analogues of the latter at the vesamicol receptor (VR) with the corresponding affinities at sigma 1 and sigma 2 binding sites. For this study, the parent structure 1 was divided into three fragments: A (cyclohexyl), B (piperidyl) and C (phenyl). Vesamicol analogues were then selected to reflect structural modifications in these fragments. Consistent with earlier reports, vesamicol was found to exhibit nanomolar affinities at the VR and sigma 1 and sigma 2 sites, resulting in poor selectivity for the VR over the sigma sites. Vesamicol analogues characterized by an acyclic A-fragment showed moderate to low affinities at the VR and moderate to high affinities at sigma 1 and sigma 2 sites. As a result, many of these analogues showed poor selectivity for the VR. Replacement of the C4 carbon of 1 with a halobenzyl amine resulted in higher affinities at the VR coupled with moderate to low affinities at sigma 1 and sigma 2 sites. The introduction of a benzofused substituent at the C4 and C5 positions of 1 (compound 2) resulted in a 200-fold increase in affinity at the VR accompanied by a 5- to 6-fold decrease in affinity at sigma 1 and sigma 2 sites relative to the parent structure. Consequently, compound 2 showed 12,000-fold higher affinity at the VR than at sigma sites. Restricting the rotation of fragment C relative to B (by means of alkyl and alkenyl bridges) generally yielded analogues with subnanomolar affinities at the VR. The corresponding affinities of these spirofused conformationally restricted analogues were moderate to poor at sigma 1 and sigma 2 sites when fragment A was preserved. In contrast, the affinities at sigma 1 and sigma 2 sites were decreased 3- to 11-fold when fragment A was modified at position C4 and decreased up to 100-fold with benzofusion at the C4 and C5 positions of fragment A. Consequently, the spirofused analogues 15-19 were among the most selective VR ligands examined. Thus, the effect of conformational restriction in fragments A and B-C is to increase affinity at the VR while decreasing affinity at sigma 1 and sigma 2 sites, and thereby increasing selectivity for the VR over the sigma sites.

Self-administration of the dopamine D3 agonist 7-OH-DPAT in rhesus monkeys is modified by prior cocaine exposure.

Cocaine self-administration was compared with responding maintained by the dopamine D3 agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) in rhesus monkeys. In the first experiment, monkeys (n = 3) with an extensive cocaine history responded under a fixed-interval (FI) 5-min schedule of IV cocaine (0.03 mg/kg per injection) presentation, during daily 4-h sessions. When responding was stable, dose-response curves were determined for cocaine (0.01-0.3 mg/kg per injection) and 7-OH-DPAT (0.001-0.1 mg/kg per injection); each dose was available for at least five consecutive sessions. When substituted for the baseline dose of cocaine, other doses of cocaine and 7-OH-DPAT maintained rates higher than responding maintained by saline injections, in all monkeys. 7-OH-DPAT maintained response rates equal to or higher than rates of cocaine-maintained responding in all monkeys. In a second experiment, acquisition of 7-OH-DPAT self-administration was evaluated in a group of cocaine-naive monkeys (n = 3). Various doses of 7-OH-DPAT (0.003-0.03 mg/kg per injection) were available under an FI 15-s schedule during daily 4-h sessions. After 10-13 sessions, 7-OH-DPAT self-administration could not be trained in any cocaine-naive monkey. When cocaine was made available to these monkeys, responding was reliably maintained within one to four sessions and the schedule was gradually increased to FI 5-min. After stable responding under an FI 5-min schedule of 0.03 mg/kg per injection cocaine presentation, 7-OH-DPAT (0.01 mg/kg per injection) was again made available to two of the monkeys, and maintained responding at rates higher than saline. To determine better whether a history of responding maintained by another reinforcer would result in high rates of 7-OH-DPAT self-administration, two cocaine-naive monkeys were trained to respond under an FI 5-min schedule of food presentation. Substituting 7-OH-DPAT (0.003-0.03 mg/kg per injection) for food resulted in very low rates of responding. Taken together, these results suggest that despite comparable reinforcing effects in cocaine-substitution studies, 7-OH-DPAT and cocaine differ in their rate of acquisition, perhaps indicating a lower abuse liability for 7-OH-DPAT.

The effects of benzamide analogues on cocaine self-administration in rhesus monkeys.

RATIONALE: Based on the differential distribution of dopamine (DA) D(3) receptors in mesolimbic regions relative to nigrostriatal regions, the hypothesis was that D(3)-selective antagonists (i.e., higher affinity at D(3)- than D(2)-receptors) would be more potent than D(2)-selective antagonists at decreasing total cocaine intake relative to disrupting rates of responding. OBJECTIVE: To evaluate the effects of acute administration of seven DA antagonists with varying affinities for D(2) and D(3) receptors in monkeys self-administering cocaine. METHODS: Rhesus monkeys were trained to self-administer intravenous cocaine (0.01-0.3 mg/kg per injection) under a fixed-interval (FI) 5-min schedule during daily 4-h sessions. The use of a FI schedule allowed for independent assessment of rate effects and changes in reinforcement frequency as a consequence of drug pretreatments. The compounds examined, in order of D(3) binding affinity, were: 2,3-dimethoxy-N-(9-p-fluorobenzyl)-azabicyclo[3.3. 1]nonan-3beta-yl benzamide (MABN) = eticlopride = 5-bromo- 2, 3-dimethoxy-N-[1-(4-fluorobenzyl)piperidin-4-yl]benz-amide (BBP) > spiperone > fluoroclebopride (FCP) > 2, 3-dimethoxy-N-(p-fluorobenzyl)piperdin-4-yl benzamide (MBP) > haloperidol. RESULTS: In the absence of any pretreatments, cocaine-maintained responding varied as a function of dose and was characterized as an inverted U-shaped function, while cocaine intake increased in a dose-related fashion. When the dose of cocaine that maintained peak rates was available, all DA antagonists decreased response rates and cocaine intake in a dose- dependent manner. Increases in cocaine dose attenuated the effects of the DA antagonists, resulting in rightward shifts of the cocaine dose-response curves. Based on the ratio of behavioral potency at decreasing response rates relative to intake (ED(50) rate/ED(50) intake) when the highest cocaine dose was available, the order of potency and ED(50) ratio values were: MABN (2.5) > eticlopride (1. 63) > BBP = spiperone (1.5) > FCP (1.35) > MBP = haloperidol (0.89). This order parallels each compound's affinity at D(3) receptors (r(2)=0.84) to a greater degree than D(2) receptor affinity (r(2)=0. 34). CONCLUSIONS: These results, using a FI schedule of cocaine self-administration, suggest that D(3) receptor antagonists are more likely to selectively decrease intake relative to response rates than D(2) receptor antagonists.