Myocyte enhancer factor 2C regulation of hepatocellular carcinoma via vascular endothelial growth factor and Wnt/ß-catenin signaling.

Hepatocellular carcinoma (HCC) is one of the leading malignancies worldwide. Myocyte enhancer factor 2C (MEF2C) was traditionally regarded as a development-associated factor and was recently reported to be an oncogene candidate. We have previously reported overexpression of MEF2C in HCC; however, the roles of MEF2C in HCC remain to be clarified. In this study, HCC cell lines and a xenograft mouse model were used to determine the functions of MEF2C in vitro and in vivo, respectively. Specific plasmids and small interfering RNA were used to upregulate and downregulate MEF2C expression, respectively. Functional assays were performed to assess the influence of MEF2C on cell proliferation, and VEGF-induced vasculogenic mimicry, migration/invasion as well as angiogenesis. Co-immunoprecipitation was conducted to identify the interaction of MEF2C and ß-catenin. Human HCC tissue microarrays were used to investigate correlations among MEF2C, ß-catenin and involved biomarkers. MEF2C was found to mediate VEGF-induced vasculogenic mimicry, angiogenesis and migration/invasion, with involvement of the p38 MAPK and PKC signaling pathways. However, MEF2C itself inhibited tumor growth in vitro and in vivo. MEF2C was upregulated by and directly interacted with ß-catenin. The nuclear translocation of ß-catenin blocked by MEF2C was responsible for MEF2C-mediated growth inhibition. The nuclear translocation of MEF2C was associated with intracellular calcium signaling induced by ß-catenin. HCC microarrays showed correlations of nuclear MEF2C with the angiogenesis-associated biomarker, CD31, and cytosolic MEF2C with the proliferation-associated biomarker, Ki-67. MEF2C showed double-edged activities in HCC, namely mediating VEGF-induced malignancy enhancement while inhibiting cancer proliferation via blockade of Wnt/ß-catenin signaling. The overall effect of MEF2C in HCC progression regulation was dictated by its subcellular distribution. This should be determined prior to any MEF2C-associated intervention in HCC.

Prohibitin as a novel target protein of luteinizing hormone in ovarian epithelial carcinogenesis.

The exact role of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in ovarian epithelial carcinoma (OEC) development has not been yet characterized. This prompted us to identify particular proteins to better understand the underlying mechanism. Total proteins from ovarian epithelial tumor (OET) cells treated with gonadotropins were analyzed by proteomics. Western blot and immunohistochemistry were used to validate the target protein (prohibitin) and to detect its expression in human ovarian tissue of serous tumors. As the results, prohibitin was found to be significantly up-regulated by LH, with a maximum of 2.5-fold increase at the concentration of 200 mIU/mL. The expression of prohibitin was steadily decreased from benign serous cystadenomas to borderline tumors and serous carcinomas (P < 0.0001). The difference between any two groups was significant (P < 0.001). Collectively, data from this study indicate that prohibitin is one LH-associated protein and it may be protective of ovarian cancer development and progression, supporting that LH may play an inhibitory role in ovarian tumorigenesis.

IBOX(2-(4'-dimethylaminophenyl)-6-iodobenzoxazole): a ligand for imaging amyloid plaques in the brain.

It is well known that overproduction and accumulation of beta-amyloid (Abeta) plaques in the brain is a key event in the pathogenesis of Alzheimer's disease (AD). Previously it was demonstrated that [125I]TZDM, 2-(4'-dimethylaminophenyl)-6-iodobenzothiazole, a thioflavin derivative, was an effective ligand with good in vitro and in vivo binding characteristics. To further improve the initial uptake and washout rate from the brain, important properties for in vivo imaging agents, a novel radioiodinated ligand, 2-(4'-dimethylaminophenyl)-6-iodobenzoxazole ([125I]IBOX, 3), for detecting Abeta plaques in the brain, was synthesized and evaluated. The new iodinated ligand, IBOX, is based on an isosteric replacement of a sulfur atom of TZDM by an oxygen, by which the molecular weight is reduced while the lipophilicity of the iodinated ligand is increased. Partition coefficients (P.C.) of these two ligands were 70 and 124 for TZDM and IBOX, respectively. In vitro binding study indicated that the isosteric displacement yielded a new ligand with equal binding potency to Abeta(1-40) aggregates (K(i) = 1.9 and 0.8 nM for TZDM and IBOX, respectively). Autoradiography of postmortem brain sections of a confirmed AD patient by [125I]IBOX showed excellent labeling of plaques similar to that observed with [125I]TZDM. More importantly, in vivo biodistribution of [125I]IBOX in normal mice displayed superior peak brain uptake (2.08% at 30 min vs 1.57% at 60 min dose/brain for [125I]IBOX and [125I]TZDM, respectively). In addition, the washout from the brain was much faster for [125I]IBOX as compared to [125I]TZDM. Based on the data presented for [125I]IBOX, it is predicted that the brain trapping of this new radioiodinated ligand in the Abeta containing regions will be more favorable than that of the parent compound, [125I]TZDM. Further evaluation of [125I]IBOX is warranted to confirm the Abeta plaque labeling properties in vivo.

The fluorescent Congo red derivative, (trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), labels diverse beta-pleated sheet structures in postmortem human neurodegenerative disease brains.

A novel Congo red-derived fluorescent probe (trans, trans),-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) that binds to amyloid plaques of postmortem Alzheimer's disease brains and in transgenic mouse brains in vivo was designed as a prototype imaging agent for Alzheimer's disease. In the current study, we used BSB to probe postmortem tissues from patients with various neurodegenerative diseases with diagnostic lesions characterized by fibrillar intra- or extracellular lesions and compared these results with standard histochemical dyes such as thioflavin S and immunohistochemical stains specific for the same lesions. These data show that BSB binds not only to extracellular amyloid beta protein, but also many intracellular lesions composed of abnormal tau and synuclein proteins and suggests that radioiodinated BSB derivatives or related ligands may be useful imaging agents to monitor diverse amyloids in vivo.

Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared.

Recently, we found that chromosome 8p deletion might be associated with hepatocellular carcinoma (HCC) metastasis by analyzing the differences in chromosomal alterations between primary tumors and their matched metastatic lesions of HCC with comparative genomic hybridization (CGH) (Qin et al. 1999). To further confirm this interesting finding, the genomic changes of two models bearing human HCC with different metastatic potentials (LCI-D20 and LCI-D35), and the new human HCC cell line with high metastatic potential (MHCC97) were analyzed by CGH. Gains on 1q, 6q, 7p, and 8q, and losses on 13p, 14p, 19p, 21, and 22 were detected in both LCI-D20 and LCI-D35 models. However, high copy number amplification of a minimum region at 1q12-q22 and 12q, and deletions on 1p32-pter, 3p21-pter, 8p, 9p, 10q, 14q, and 15p were detected only in the LCI-D20 model. Gains on 1p21-p32, 2p13-p21, 6p12-pter, 9p, 15q, and 16q11-q21, and losses on 2p23-pter, 4q24-qter, 7q31-qter, 12q, 17p, and 18 were detected only in the LCI-D35 model. The chromosomal aberration patterns in the MHCC97 cell line were similar to its parent LCI-D20 model, except that gains on 19q and losses on 4, 5, 10q, and 13q were found only in the cell line. These results provide some indirect clues to the metastasis-related chromosomal aberrations of HCC and further support the finding that 8p deletion is associated with HCC metastasis. 1q12-22 and 12q might harbor a novel oncogene(s) that contributes to the development and progression of HCC. Amplification on 8q and deletions on 4q and 17p may be not necessary for HCC metastasis.

Isomerization of (Z,Z) to (E,E)1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene in strong base: probes for amyloid plaques in the brain.

In developing probes for detecting beta-amyloid (Abeta) plaques in the brain of Alzheimer's disease (AD), we have synthesized 1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (5, BSB). Due to the presence of two double bonds, formation of four different isomers is possible. Four isomers, E,E-5, E,Z-5, Z,E-5, and Z,Z-5, were prepared. Surprisingly, all showed strong fluorescent labeling of Abeta plaques in the brain of postmortem brain sections of patients with confirmed AD. In vitro binding assay also showed that all four isomers of BSB (E,E-5, E,Z-5, Z,E-5, and Z,Z-5) displayed a similar high binding affinity inhibiting the binding of [(125)I]E,E-6, 1-iodo-2,5-bis-(3-hydroxycarbonyl-4-methoxy)styrylbenzene (IMSB) to Abeta(1-40) aggregates. The inhibition constants (K(i)) of E,E-5, E,Z-5, Z,E-5, and Z,Z-5 were 0.11 +/- 0.01, 0.19 +/- 0.03, 0.27 +/- 0.06, and 0.13 +/- 0.02 nM, respectively. Due to the fact that geometric stability of these styrylbenzenes is unknown, and the conversion of Z,Z-5 to E,E-5 may occur automatically in the binding or labeling assaying conditions, we have investigated the kinetics of conversion of Z,Z-5 to E,E-5 by NMR in D(2)O/NaOD at elevated temperatures (70, 95, and 115 degrees C). The activation energy was determined to be 14.15 kcal/mol. The results strongly suggest that the isomeric conversion at room temperature in aqueous buffer solution is unlikely. All of the styrylbenzene isomers clearly showed potential as useful tools for studying Abeta aggregates in the brain. The data suggest that, despite the rigidity of this series of styrylbenzenes, the binding sites on Abeta aggregates may have certain flexibility and the binding pockets could be adaptable for binding to other smaller ligands. Such information could be exploited to develop new ligands for detecting amyloid plaques in AD.

Radioiodinated styrylbenzenes and thioflavins as probes for amyloid aggregates.

We report for the first time that small molecule-based radiodiodinated ligands, showing selective binding to Abeta aggregates, cross the intact blood-brain barrier by simple diffusion. Four novel ligands showing preferential labeling of amyloid aggregates of Abeta(1-40) and Abeta(1-42) peptides, commonly associated with plaques in the brain of people with Alzheimer's disease (AD), were developed. Two 125I-labeled styrylbenzenes, (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene, 12 (ISB), and (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene, 13 (IMSB), and two 125I-labeled thioflavins, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 18a (TZDM), and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, 18b (TZPI), were prepared at a high specific activity (2200 Ci/mmol). In vitro binding studies of these ligands showed excellent binding affinities with Kd values of 0.08, 0.13, 0.06, and 0.13 nM for aggregates of Abeta(1-40) and 0.15, 0.73, 0.14, and 0.15 nM for aggregates of Abeta(1-42), respectively. Interestingly, under a competitive-binding assaying condition, different binding sites on Abeta(1-40) and Abeta(1-42) aggregates, which are mutually exclusive, were observed for styrylbenzenes and thioflavins. Autoradiography studies of postmortem brain sections of a patient with Down's syndrome known to contain primarily Abeta(1-42) aggregates in the brain showed that both [(125)I]18a and [125I]18b labeled these brain sections, but [125I]13, selective for Abeta(1-40) aggregates, exhibited very low labeling of the comparable brain section. Biodistribution studies in normal mice after an iv injection showed that [125I]18a and [(125)I]18b exhibited excellent brain uptake and retention, the levels of which were much higher than those of [125I]12 and [125I]13. These findings strongly suggest that the new radioiodinated ligands, [125I]12 (ISB), [125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI), may be useful as biomarkers for studying Abeta(1-40) as well as Abeta(1-42) aggregates of amyloidogenesis in AD patients.

Identification of novel regions of allelic loss from a genomewide scan of esophageal squamous-cell carcinoma in a high-risk Chinese population.

Esophageal cancer is one of the most common fatal cancers worldwide. Deletions of genomic regions are thought to be important in esophageal carcinogenesis. We conducted a genomewide scan for regions of allelic loss using microdissected DNA from 11 esophageal squamous-cell carcinoma patients with a family history of upper gastrointestinal tract cancer from a high-risk region in north central China. Allelic patterns of 366 fluorescently labeled microsatellite markers distributed at 10-cM intervals over the 22 autosomal chromosomes were examined. We identified 14 regions with very high frequency (>/= 75%) loss of heterozygosity (LOH), including broad regions encompassing whole chromosome arms (on 3p, 5q, 9p, 9q, and 13q), regions of intermediate size (on 2q, 4p, 11p, and 15q), and more discrete regions identified by very high frequency LOH for a single marker (on 4q, 6q, 8p, 14q, and 17p). Among these 14 regions were 7 not previously described in esophageal squamous-cell carcinoma as having very high frequency LOH (on 2q, 4p, 4q, 6q, 8p, 14q, and 15q). The very high frequency LOH regions identified here may point to major susceptibility genes, including potential tumor suppressor genes and inherited gene loci, which will assist in understanding the molecular events involved in esophageal carcinogenesis and may help in the development of markers for genetic susceptibility testing and screening for the early detection of this cancer. Genes Chromosomes Cancer 27:217-228, 2000. Published 2000 Wiley-Liss, Inc.

Single-photon emission tomography imaging of serotonin transporters in the nonhuman primate brain with [(123)I]ODAM.

We have described previously a selective serotonin transporter (SERT) radioligand, [(123)I]IDAM. We now report a similarly potent, but more stable IDAM derivative, 5-iodo-2-[2-[(dimethylamino)methyl]phenoxy]benzyl alcohol ([(123)I]ODAM). The imaging characteristics of this radioligand were studied and compared against [(123)I]IDAM. Dynamic sequences of single-photon emission tomography (SPET) scans were obtained on three female baboons after injection of 375 MBq of [(123)I]ODAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 120 min after injection of [(123)I]ODAM. Total integrated brain uptake of [(123)I]ODAM was about 30% higher than [(123)I]IDAM. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT. Peak specific binding in the midbrain occurred 120 min after injection, with an equilibrium midbrain to cerebellar ratio of 1. 50+/-0.08, which was slightly lower than the value for [(123)I]IDAM (1.80+/- 0.13). Both the binding kinetics and the metabolism of [(123)I]ODAM were slower than those of [(123)I]IDAM. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited washout from areas with high concentrations of SERT, with a dissociation kinetic rate constant k(off)=0.0085+/-0.0028 min(-1) in the midbrain. Similar studies using nisoxetine and methylphenidate showed no displacement, consistent with its low binding affinity to norepinephrine and dopamine transporters, respectively. These results suggest that [(123)I]ODAM is suitable for selective SPET imaging of SERT in the primate brain, with higher uptake and slower kinetics and metabolism than [(123)I]IDAM, but also a slightly lower selectivity for SERT.

Neutral and stereospecific Tc-99m complexes: [99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT).

Technetium-99m-labeled radiopharmaceuticals are currently the most commonly used agents in nuclear medicine. To prepare binding site-specific small molecules containing a Tc-99m complexing core, it is important to consider a ligand system, which selectively forms only one stereoisomer. A novel series of bisaminoethanethiol (BAT) derivatives as a model system were prepared. Stereoisomers of N-benzyl-3,4-di(N-2-mercaptoethyl)-amino pyrrolidines (P-BAT): (3R,4R)-P-BAT (R,R-4) and (3,4)meso-P-BAT (8), the trans and meso isomer, respectively, as a chelating group were prepared successfully. The desired Tc-99m P-BAT complexes were obtained by using Sn(II)/glucoheptonate as the reducing agent for [99mTc]pertechnetate. As predicted, after complexation with [99mTc]Tc'O, the trans isomer, (3R,4R)-P-BAT (R,R-4), showed only one isomer; whereas the corresponding meso isomer, (3,4)meso-P-BAT (8), produced two distinctive complexes isolated readily by high performance liquid chromatography (HPLC). The [99mTc](R,S) meso-P-BAT (8) isomers showed a different lipophilicity (partition coefficient [P.C.] = 54.3 and 55.4 for peak A and peak B, respectively), as compared with that of the corresponding [99mTc](3R,4R)-P-BAT (R,R-4), trans isomer (P.C. = 163). Results of the biodistribution study in rats of these isomers show different heart and brain uptake, suggesting that the intrinsic differences in biodistribution are due to structural and stereospecific factors. Examples in this report confirm that it is possible to design stereospecific Tc-99m complexes based on the bisaminoethanethiol (N2S2, BAT) ligand system. Consideration on stereoselectivity of site-specific agents labeled with Tc-99m is likely an essential requirement on developing binding-site specific radiopharmaceuticals.

99mTc-labeled MIBG derivatives: novel 99mTc complexes as myocardial imaging agents for sympathetic neurons.

Radioactive-iodine-labeled meta-iodobenzylguanidine (MIBG) is currently being used as an in vivo imaging agent to evaluate neuroendocrine tumors as well as the myocardial sympathetic nervous system in patients with myocardial infarct and cardiomyopathy. It is generally accepted that MIBG is an analogue of norepinephrine and its uptake in the heart corresponds to the distribution of norepinephrine and the density of sympathetic neurons. A series of MIBG derivatives containing suitable chelating functional groups N2S2 for the formation of [TcvO]3+N2S2 complex was successfully synthesized, and the 99mTc-labeled complexes were prepared and tested in rats. One of the compounds, [99mTc]2, tested showed significant, albeit lower, heart uptakes post iv injection in rats (0.21% dose/g at 4 h) as compared to [125I]MIBG (1.7% dose/g at 4 h). The heart uptake of the 99mTc-labeled complex appears to be specific and can be reduced by co-injection with nonradioactive MIBG or by pretreatment with desipramine, a selective norepinephrine transporter inhibitor. Further evaluation of the in vitro uptake of [99mTc]2 in cultured neuroblastoma cells displayed consistently lower, but measurable uptake (approximately 10% of that for [125I]MIBG). These preliminary results suggested that the mechanisms of heart uptake of [99mTc]2 may be related to those for [125I]MIBG uptake. If suitable 99mTc-labeled MIBG derivatives can be further developed, the prevalent availability of 99mTc in nuclear medicine clinics will allow them to be readily available for widespread application.

Isoindol-1-one analogues of 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridyl)-p-iodobenzamido]ethyl]pipera zine (p-MPPI) as 5-HT1A receptor ligands.

In developing radioiodinated antagonists for in vivo imaging of 5-HT1A receptors with SPECT, a series of new arylpiperazine benzamido derivatives, including 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridyl)-p-iodobenzamido]ethyl]p iperazine (p-MPPI, 31) (Kd = 0.36 nM), as potential ligands for 5-HT1A receptors were reported previously. However, rapid in vivo metabolism may have caused the breakdown of the amide bond of [123I]-31 and rendered this agent obsolete as an in vivo imaging agent in humans. To improve the in vivo stability of 31, a series of cyclized amide analogues were designed and synthesized. In vitro binding, metabolic stability, and in vivo biodistribution of these new derivatives were investigated. Several five-membered-ring isoindol-1-ones displayed very high in vitro binding affinity, especially 2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-6-nitro-3-phenyl-2, 3-dihydroisoindol-1-one, 15, 3-hydroxy-6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}- 3- phenyl-2,3-dihydroisoindol-1-one, 18, and 6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-3-phenyl-2, 3-dihydroisoindol-1-one, 21, which showed Ki values of 0.05, 0.65, and 0.07 nM, respectively. The affinities for 5-HT1A receptors of other cyclized amide derivatives, 5-(4-bromophenyl)-1-{2-[4-(2-methoxyphenyl)- piperazin-1-yl]ethyl}pyrrolidin-2-one, 25, 5-(4-iodophenyl)-1-{2-[4-(2-methoxyphenyl)piperazin- 1-yl]ethyl}pyrrolidin-2-one, 27, and 2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-2,3-dihydro- isoindol-1-one, 29, were 1.09, 2.54, and 14.9 nM, respectively. Compared to [125I]-31, iodinated cyclized amide derivatives [125I]-21 and [125I]-27 displayed a slower metabolism in human liver microsomal and cytosolic preparations. Biodistribution of [125I]-21 and [125I]-27 in rats (after an i.v. injection) displayed moderate to low brain uptakes with little or no specific localization in hippocampal region, where 5-HT1A receptors are concentrated. These data indicate that the new iodinated ligands showed high binding affinities and better metabolic stability but displayed unexpectedly low selective binding to 5-HT1A receptors in vivo. Additional structural modifications may be needed to correct the unfavorable properties displayed for these iodinated cyclized amide derivatives for in vivo biodistribution in rats.

The 5-HT1A receptor antagonist p-MPPI blocks responses mediated by postsynaptic and presynaptic 5-HT1A receptors.

The present experiments examined the ability of the novel 5-HT1A receptor antagonist to block responses mediated by postsynaptic and presynaptic 5-HT1A receptors in vivo. Pretreatment with p-MPPI reduced or blocked the effect of the 5-HT1A receptor agonist 8-OH-DPAT on two responses mediated by postsynaptic 5-HT1A receptors, reduction of body temperature and the 5-HT behavioral syndrome. Administration of p-MPPI alone did not alter body temperature or produce symptoms of the 5-HT syndrome. Pretreatment with p-MPPI also blocked the ability of 8-OH-DPAT to reduce extracellular 5-HT in the striatum, a response mediated by presynaptic 5-HT1A receptors in the dorsal raphe nucleus, but did not alter striatal 5-HT when administered alone. These results indicate that p-MPPI is an effective 5-HT1A receptor antagonist in vivo with no intrinsic activity. p-MPPI may prove to be a useful pharmacological tool for studying 5-HT1A receptors and their involvement in anxiety and affective disorders.

P-[18F]-MPPF: a potential radioligand for PET studies of 5-HT1A receptors in humans.

The purpose of this study was to develop a radiopharmaceutical that could be used to selectively image 5-HT1A receptors with positron emission tomography (PET). No-carrier-added 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-[18F] fluorobenzamido]ethylpiperazine (p-[18F]-MPPF, 2) was synthesized by the nucleophilic substitution of the corresponding nitro precursor 1 with K[18F]/Kryptofix 2.2.2. in dimethyl sulfoxide (DMSO) at 140 degrees C for 20 min followed by purification with high-performance liquid chromatography (HPLC) in 10% yield in a synthesis time of 90 min from end of bombardment (EOB). Specific activity was 1-4 Ci/microM. Biodistribution studies in rats showed that the initial uptake of 2 in the brain was high (0.7% dose/g tissue at 2 min). It was then rapidly eliminated. Rates of elimination were significantly slower in brain regions with high concentrations of 5-HT1A receptors (hippocampus, cortex, and hypothalamus) than in control regions. The maximum hippocampal/cerebellar ratio was 5.6:1 at 30 min postinjection. Uptake values in serotonergic, but not in control, regions were significantly reduced by prior treatment with either (+/-)-8-OH-DPAT (2 mg/kg, i.v., 5 min prior) or WAY 100635 (1 mg/kg, i.v., 5 min prior). Radioactivity in the femur did not increase with time, suggesting that in vivo defluorination may not be the major route of metabol sm. PET studies of 2 in a monkey demonstrated selective uptake and retention of 2 in the hippocampus. The hippocampal/cerebellar ratio was 3:1 at 30 min postinjection. The ratio was reduced to 1:1 by administering (+/-)-8-OH-DPAT (2 mg/kg, i.v.) 23 min postinjection of 2. Analyses of arterial plasma by HPLC revealed that 20% of radioactivity in the plasma remained as the parent compound 2 at 30 min postinjection. The results suggest that p-[18F]-MPPF may be a useful radioligand for studying cerebral 5-HT1A receptors in humans with PET techniques.

Characterization of a novel iodinated ligand, IPMPP, for human dopamine D4 receptors expressed in CHO cells.

A novel radioiodinated ligand with a high specific activity (2,200 Ci/mmol), 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b)pyridine ([125I]IPMPP), was successfully prepared. Binding characteristics of [125I]IPMPP were evaluated using human dopamine D4 (D4.2 variant) receptors expressed in Chinese hamster ovary (CHO) cells. Saturation analysis revealed high-affinity binding sites for [125I]IPMPP (Kd = 0.39 +/- 0.18 nM). The number of D4 receptors labeled with [125I]IPMPP at room temperature was four times higher than that labeled with [125I]S(-)5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol/mg protein vs. 125 fmol/mg protein). A significant decrease in the number of binding sites was observed with [125I]S(-)5-OH-PIPAT when assays were carried out at a higher temperature (37 degrees C vs. 25 degrees C). In contrast to [125I]S(-)5-OH-PIPAT, [125I]IPMPP labeled more D4 sites at 37 degrees C. Neither magnesium ion nor guanylimidodiphosphate (Gpp(NH)p) affected [125I]IPMPP binding. These data support the conclusion that [125I]IPMPP is an antagonist ligand. The potency of various compounds, including clozapine, to inhibit [125I]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors. In addition, measuring D4 receptor stimulation of [35S]GTPgammaS binding further demonstrated the antagonist property of IPMPP.

In vivo SPECT imaging of 5-HT1A receptors with [123I]p-MPPI in nonhuman primates.

The in vivo imaging of a novel iodinated phenylpiperazine derivative for 5-HT1A receptors, [123I]p-MPPI (4-(2'-methoxy-)phenyl-1-[2'-(n-2"-pyridinyl)-p-iodobenzamido-] ethyl-piperazine), using single photon emission computed tomography (SPECT), was evaluated in nonhuman primates. After an i.v. injection, [123I]p-MPPI penetrated the blood-brain barrier quickly and localized in brain regions where 5-HT1A receptor density is high (hippocampus, frontal cortex, cingulate gyrus, entorhinal cortex). Maximum ratio of hippocampus to cerebellum was 3 to 1 at 50 min postinjection. The specific binding of the radioligand in the hippocampal region, an area rich in 5-HT1A receptor density, was blocked by a chasing dose of (+/-) 8-OH-DPAT (2 mg/kg, i.v.) or non-radioactive p-MPPI (1 mg/kg, i.v.), whereas the regional distribution of [123I]p-MPPI was unaffected by treatment with non 5-HT1A agents, such as ketanserin. Ex vivo and in vitro autoradiographic studies using monkey brain further confirmed that the specific binding of [123I]p-MPPI is associated with 5-HT1A receptor sites. However, the initial attempt at [123I]p-MPPI human imaging studies did not display specific localization of 5-HT1A receptors. This discrepancy observed for [123I]p-MPPI may be due to a dramatic difference in metabolic pathways between humans and monkeys.

New 5-HT1A receptor antagonist: [3H]p-MPPF.

A new 5-HT1A receptor antagonist ligand, [3H]p-MPPF, 4-(2'-methoxy-)-phenyl-1-[2'-(N-2"-pyridyl)-p-fluorobenzamido] ethyl-piperazine, was prepared and characterized. It demonstrated high affinity and selectivity toward 5-HT1A receptors (Kd = 0.34 +/- 0.12 nM and Bmax = 145 +/- 35 fmol/mg protein in rat hippocampal membrane homogenates). The binding is not sensitive to 100 microM Gpp(NH)p. Initial autoradiography studies of rat brain sections exhibit regional localization consistent with the known 5-HT1A receptor distribution. This potential 5-HT1A antagonist ligand may provide a powerful tool for 5-HT1A receptor pharmacology studies in the central nervous system.

NCS-MPP (4-(2'-methoxy-phenyl)-1-[2'-(N-2"-pyridyl)-p-isothiocyanobenz amido]-ethyl-piperazine): a high affinity and irreversible 5-HT1A receptor ligand.

A novel irreversible 5-HT1A receptor binding ligand, NCS-MPP (4-(2'- methoxy-phenyl)-1-[2'-(N-2"-pyridyl)-p-isothiocyanobenzamido]- ethyl-piperazine), based on the new 5-HT1A receptor antagonist p-MPPI (4-(2'-methoxy-phenyl)-1-[2'-(N-2"-pyridyl)-p-iodobenzamido]-ethyl -piperazine ), was synthesized, and its binding characteristics were evaluated using in vitro homogenate binding with rat hippocampal membranes. The Ki value of NCS-MPP was estimated to be 1.8 +_ 0.2 nM using analysis of concentration-dependent inhibition for the binding of [125I]p-MPPI to 5-HT1A receptors. NovaScreen of NCS-MPP showed low to moderate binding affinities to alpha-1, alpha-2-adrenergic and 5-HT2 receptors, with Ki values of 350, 420, and 103 nM, respectively. These data strongly suggest that the ligand bound to 5-HT1A receptors with high affinity and high selectivity. Irreversible inhibition of [125I]p-MPPI binding by NCS-MPP following a 5 min incubation at room temperature was concentration dependent; the inhibition increased to 50% at a concentration less than 10 nM, and became more pronounced (90%) at 400 nM. Under similar assay conditions, NCS-MPP was significantly less efficient in irreversibly inhibiting agonist ligand [125I]8-OH-PIPAT binding to 5-HT1A receptors at lower concentrations (<10nM). After pretreatment of membranes with a low concentration of NCS-MPP (2nM), there was an apparent loss of [125I]p-MPPI binding sites, as expected, but no change in the binding affinity (Kd) was observed. However, the significant increase in Kd at a higher concentration of NCS-MPP (50 nM) indicated that there may be a secondary alkylation site, which may not be directly involved in p-MPPI binding to receptors; nevertheless, it would lead to an increased Kd value. The availability of an irreversible ligand, NCS-MPP, may provide a useful tool for studies of 5-HT1A receptors in the central nervous system.

4-(2'-Methoxy-phenyl)-1-[2'-(n-2"-pyridinyl)-p-iodobenzamido]-ethyl- piperazine ([125I]p-MPPI) as a new selective radioligand of serotonin-1A sites in rat brain: in vitro binding and autoradiographic studies.

Binding characteristics of a radioiodinated serotonin-1A (5-HT1A) receptor antagonist, 4-(2'-methoxy-phenyl)-1-[2'-(n-2"-pyridinyl)-p- iodobenzamido]-ethyl-piperazine ([125I]p-MPPI) were evaluated using in vitro homogenate binding and autoradiographic techniques in rat brains. [125I]p-MPPI displayed a Kd value of 0.32 +/- 0.04 nM (in the presence of MgCl2) and a Bmax value of 315 +/- 60 fmol/mg of protein in rat hippocampal homogenates. The number of 5-HT1A receptors labeled by [125I]p-MPPI was 40% higher than that labeled by trans-8-hydroxy-2-(N-n-propyl-N-3'-iodo-2'- propenyl)aminotetralin ([125I]R(+)8-OH-PIPAT) (225 +/- 47 fmol/mg of protein), a radioiodinated 5-HT1A agonist. The magnesium ion showed an inhibitory effect on [125I]p-MPPI binding but increased the specific binding of [125I]R(+)8-OH-PIPAT. A significant increase in Bmax values in the presence of guanyl nucleotides was observed for [125I]p-MPPI (control, 307 +/- 35 fmol/mg of protein; with GTP, 345 +/- 30 fmol/mg of protein; with guanylyl-imidodiphosphate, 362 +/- 35 fmol/mg of protein); however, both guanyl nucleotides significantly reduced the Bmax values measured by [125I]R(+)8-OH-PIPAT (control, 213 +/- 50 fmol/mg of protein; with GTP, 133 +/- 20 fmol/mg of protein; with guanylyl-imidodiphosphate, 108 +/- 20 fmol/mg of protein). The binding characteristics of [125I]p-MPPI for 5-HT1A receptors suggest that p-MPPI is an antagonist for 5-HT1A receptors. In vitro autoradiographic studies in rat brain sections with [125I]p-MPPI showed specific labeling of areas rich in 5-HT1A receptors and the regional distribution closely matched those labeled by [125I]R(+)8-OH-PIPAT.(ABSTRACT TRUNCATED AT 250 WORDS)