Synthesis and Biological Evaluation of 99mTc-Labeled Phenylpiperazine Derivatives as Selective Serotonin-7 Receptor Ligands for Brain Tumor Imaging.

With a poor prognosis, glioblastoma multiforme is the most aggressive tumor of the central nervous system in humans. The aim of this study was to develop novel tracers for the tumor targeting and imaging of overexpressed serotonin-7 receptors (5-HT7Rs) in U-87 MG glioma xenografted nude mice. Two phenylpiperazine derivatives named as PHH and MPHH were designed, and the corresponding radiotracers 99mTc-PHH and 99mTc-MPHH were synthesized in high radiochemical purity (>95%). 99mTc-MPHH showed a higher affinity to 5-HT7Rs on U-87 MG cells compared to 99mTc-PHH. In biodistribution studies, the radiocomplexes showed good brain uptake at 15 min combined with good radioactivity retention in the brain for 240 min. Regional rabbit brain studies indicated a higher radioactivity concentration in the hippocampus and diencephalon than in the cerebellum. Compared to 99mTc-MPHH, the 99mTc-PHH exhibited a significantly increased tumor uptake at 15 and 60 min, but the rapid blood clearance of 99mTc-MPHH led to enhanced tumor-to-muscle ratios at 240 min. A significant reduction in tumor uptake 60 min after an injection of pimozide (5-HT7 receptor antagonist) confirms the tumor uptake was receptor-mediated specifically. The tumor-to-contralateral muscle tissue ratio of 99mTc-PHH and 99mTc-MPHH in nude mice with U-87 MG xenograft was measured (5.25 and 4.65) at 60 min as well as (6.25 and 6.76) at 240 min, respectively.

The Novel Small Molecule TRVA242 Stabilizes Neuromuscular Junction Defects in Multiple Animal Models of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder in which the neuromuscular junction progressively degenerates, leading to movement difficulties, paralysis, and eventually death. ALS is currently being treated by only two FDA-approved drugs with modest efficacy in slowing disease progression. Often, the translation of preclinical findings to bedside terminates prematurely as the evaluation of potential therapeutic compounds focuses on a single study or a single animal model. To circumscribe these issues, we screened 3,765 novel small molecule derivatives of pimozide, a recently identified repurposed neuroleptic for ALS, in Caenorhabditis elegans, confirmed the hits in zebrafish and validated the most active compounds in mouse genetic models. Out of the 27 small molecules identified from the high-throughput screen in worms, 4 were found to recover locomotor defects in C. elegans and genetic zebrafish models of ALS. TRVA242 was identified as the most potent compound as it significantly improved efficiency in rescuing locomotor, motorneuron, and neuromuscular junction synaptic deficits in a C. elegans TDP-43 model and in multiple zebrafish genetic (TDP-43, SOD1, and C9ORF72) models of ALS. The actions of TRVA242 were also conserved in a mammalian model as it also stabilized neuromuscular junction deficits in a mouse SOD1 model of ALS. Compounds such as TRVA242 therefore represent new potential therapeutics for the treatment of ALS.

ID1 Is Critical for Tumorigenesis and Regulates Chemoresistance in Glioblastoma.

Glioblastoma is the most common primary brain tumor in adults. While the introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid tumor recurrence remains universal. The transcriptional regulatory protein, inhibitor of DNA-binding-1 (ID1), is a key regulator of cell phenotype in cancer. We show that CRISPR-mediated knockout of ID1 in glioblastoma cells, breast adenocarcinoma cells, and melanoma cells dramatically reduced tumor progression in all three cancer systems through transcriptional downregulation of EGF, which resulted in decreased EGFR phosphorylation. Moreover, ID1-positive cells were enriched by chemotherapy and drove tumor recurrence in glioblastoma. Addition of the neuroleptic drug pimozide to inhibit ID1 expression enhanced the cytotoxic effects of temozolomide therapy on glioma cells and significantly prolonged time to tumor recurrence. Conclusively, these data suggest ID1 could be a promising therapeutic target in patients with glioblastoma. SIGNIFICANCE: These findings show that the transcriptional regulator ID1 is critical for glioblastoma initiation and chemoresistance and that inhibition of ID1 enhances the effect of temozolomide, delays tumor recurrence, and prolongs survival.

Evaluation of the guinea pig monophasic action potential (MAP) assay in predicting drug-induced delay of ventricular repolarisation using 12 clinically documented drugs.

INTRODUCTION: While the dog in vivo model is commonly employed in the later phase of discovery for assessing drug-induced QT prolongation, an early screening assay is valuable when selecting compounds for further development and when compound availability usually is low. One such screening assay is the anaesthetised guinea pig monophasic action potential (MAP) model. The aim of the present study was to evaluate the ability of this model to detect proarrhythmic properties by testing a set of reference compounds with known clinical profile. Moreover, these results were compared to data previously obtained using in vivo canine QT assays (QT PRODACT study). METHODS: Anaesthetised and ventilated male guinea pigs were vagotomised and pretreated with propranolol. After thoracotomy, a pacing electrode was clipped to the left atrial appendage and a suction MAP electrode positioned on the left ventricular epicardium. The drug or corresponding vehicle was injected intravenously in cumulative doses and MAP duration at 90% repolarisation (MAPD90) was recorded during cardiac pacing. RESULTS: The 8 drugs known to be proarrhythmic in the clinic all displayed dose-dependent prolongation of MAPD90, while the 4 drugs devoid of dysrhythmia in man had no effect. When comparing doses producing a 10% MAPD90 increase with doses reported to increase QTc by 10% in dogs a strong correlation was seen (R(2) 0.94 and 0.58 for anaesthetised and conscious dogs, respectively). DISCUSSION: The guinea pig MAP assay identified all clinically positive drugs while negative drugs were without effect on ventricular repolarisation. Furthermore, a good concurrence is shown between the guinea pig and dog models in identifying compounds with proarrhythmic properties. Overall, the study reinforces the anaesthetised guinea pig MAP model as a reliable assay predicting QT liability of new chemical entities and as a highly sensitive early screening model for cardiovascular risk.

Progesterone improves the number and quality of hormone induced Fowler toad (Bufo fowleri) oocytes.

Combinations of progesterone, lutenizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotrophin (hCG), and the dopamine-2 (DA2) receptor antagonist 1-[1-[4,4-bis(4-Fluorophenyl)butyl]-4-piperidinyl]-1,3-dihydro-2H-benzimidazol-2-one (Pimozide; Orap) were tested for improvement of spawning rates, oocyte numbers, fertilization and neurulation rates of the Fowler toad (Bufo fowleri). Only treatments combined with progesterone produced large numbers of oocytes. The best treatment on oocyte numbers, neurulation rates, and the number of neurulas was with 5 mg progesterone, 20 mic.g LHRHa, and 0.25 mg Pimozide. Progesterone (5 mg) with 60 mic.g LHRHa gave high spawning rates, oocyte numbers, and fertilization rates but neurulation rates were low. Progesterone alone in high repeated doses did not result in ovulation. High doses of LHRHa (60 mic.g) with hCG, progesterone, and Pimozide gave the greatest number of toads spawning, however, they resulted in low oocyte numbers, fertilization and neurulation rates. A low dose of LHRHa (4 mic.g) with hCG, or hCG alone as a second administration, and progesterone with Pimozide produced few good quality oocytes. Toads were given normal ovulatory doses of hormones 24 or 48 hrs after their initial dose, but these resulted in low oocyte numbers followed by poor fertilization. Overall, these results suggest that progesterone with a dose between 20 mic.g and 60 mic.g of LHRHa may be optimal for the induction of ovulation in these toads. Moreover, Pimozide can supplement low doses of LHRHa but not replace it.

Bait-delivered pimozide causes precocious embryo implantation in mink: a fertility control option for the exotic stoat?

Stoats (Mustela erminea), an exotic pest in New Zealand, threaten the conservation of several ground-nesting bird species and broad-scale methods for their control are sought. Females are seasonally monestrous, show a 9-month period of obligatory diapause and usually do not breed more than once in their lives. A bait-delivered agent that terminates diapause and results in a non-viable embryo may have a significant impact on their reproductive success. Prolactin (PRL) is hypothesised to be the only gonadotrophin required for renewal of luteal activity and blastocyst implantation in some mustelids. We investigated the effects of bait-delivered dopamine (DA) antagonists (which stimulate the release of PRL) using a mink model (Mustela vison), a species that maintains a short period of diapause. A bait dose of 0.8 mg kg(-1) of pimozide was more effective in elevating PRL levels than equivalent doses of fluphenazine, sulpiride (P < 0.01) or haloperidol (P < 0.05). Bait doses of 1.6 mg kg(-1) pimozide given at Days 0, 3, 9 and 11 after mating caused a significant reduction in the length of pregnancy compared with a positive control and placebo (46 days v. 51 days), indicating early termination of diapause (P < 0.01). Pimozide doses caused higher elevations in PRL concentration relative to the oral placebo by Day 12, but mean PRL levels fell below all other groups by Day 18. A borderline significant increase in progesterone (P4) secretion compared with the oral placebo was detected at Day 18. These results suggest that bait-delivered pimozide can elevate PRL outside of the normal breeding season and doses of 1.6 mg kg(-1) are effective in terminating embryonic diapause in mink. The implications and limitations of these data are discussed with reference to the use of bait-delivered DA antagonists as a possible means to affect the reproductive success of wild stoats.

Increased lethality of calmodulin antagonists and bleomycin to human bone marrow and bleomycin-resistant malignant cells.

The effect of bleomycin and calmodulin antagonists on human cells was studied using a clonogenic assay. A 1-h exposure to nontoxic concentrations of the calmodulin antagonists melittin (0.5 microM), pimozide (5 microM), and chlorpromazine (20 microM) increased the lethality of bleomycin to human ovarian carcinoma cells (SK-OV). No increase was seen with chlorpromazine sulfoxide, which lacks calmodulin antagonistic activity. Maximum enhancement of bleomycin lethality by calmodulin antagonists was seen when the antagonist was present simultaneously with bleomycin rather than before or after bleomycin. The cytotoxicity of bleomycin to A-253 head and neck squamous carcinoma cells, which were 10-fold more sensitive to bleomycin alone compared to SK-OV cells, was not markedly altered by the presence of 20 microM chlorpromazine. Chlorpromazine, melittin, or pimozide also increased the toxicity of bleomycin to human granulocyte/macrophage and erythroid stem cell colonies. These results demonstrate that calmodulin antagonists can significantly increase the lethality of bleomycin to some but not all human tumor cells and that nonmalignant hematological human cells may also be affected by this combination.

Comparison of methods to improve induction of spermiation in wild-caught carp (Cyprinus carpio carpio), a threatened species from the Caspian Sea basin.

Wild carp (Cyprinus carpio carpio) forms the basis of an important fishery in the Southern Caspian Sea Basin which is increasingly underpinned by the release of cultured juveniles. A significant bottleneck to hatchery-rearing of juveniles is the spermiation of male broodstock. Therefore, four approaches to improving spermiation were investigated. The effectiveness of two delivery methods for the sustained release of salmon gonadotropin releasing hormone analogue (sGnRHa; i.e., via intramuscular cholesterol pellet vs emulsion injection) on the spermiation success and duration, sperm quality and quantity over 14days in wild-caught carp were compared to single injection of sGnRHa with Pimozide(®) (Linpe method) or carp pituitary extract (CPE). The consequence of the spermiation treatments on resulting embryonic quality was evaluated for subsequent fertilization and hatching success from wild male carp (mean weight±S.D. 1021±112g). All hormonal treatments, except for Linpe method, led to 100% spermiation of treated fish compared to only 25% in the control with no hormone intervention. The duration of spermiation, as well as the various quantitative variables of the sperm and the mean total sperm production were all generally improved with the sustained hormone delivery compared with the acute treatments. The GnRHa-FIA was the most effective method for improving spermiation.

The antipsychotic drug pimozide inhibits cell growth in prostate cancer through suppression of STAT3 activation.

Currently, drug discovery and development for clinical treatment of prostate cancer has received increased attention, specifically the STAT3 inhibitor. Our previous study reported that the neuroleptic drug pimozide had antitumor activity against hepatocellular carcinoma cells or stem-like cells through suppressing the STAT3 activity. In the present study we demonstrate that pimozide inhibits cell growth and cellular STAT3 activation in prostate cancer cells. Our results showed that pimozide inhibited prostate cancer cell proliferation in a dose- and time-dependent manner by inducing G1 phase cell cycle arrest, downregulated the ability of colony formation and sphere forming, as well as suppressed cells migration in both DU145 and LNCaP cells. Surprisingly, pimozide reduced the basal expression of phosphorylation STAT3 at tyrosine 705 and reversed the expression of phosphorylation of STAT3 induced by IL-6 addition, suggesting that pimozide can suppress cellular STAT3 activation. Thus, the antipsychotic agent pimozide may be a potential and novel therapeutic for patients with advanced prostate cancer.

Serum calcium and magnesium levels in schizophrenia. II. Possible relationship to extrapyramidal symptoms.

The relationship between serum calcium and magnesium levels and neuroleptic-induced extrapyramidal symptoms (EPS) was studied in schizophrenic patients. The 16 patients in whom EPS developed had a significantly lower mean drug-free calcium level than the six patients in whom EPS did not develop. In patients in whom EPS developed, drug-free serum calcium and magnesium levels together correlated significantly with the neuroleptic dosage at which EPS first developed; lower calcium and magnesium values predicted EPS at lower dosages. We have previously shown that both serum calcium and magnesium levels were significantly lower during neuroleptic treatment than in the drug-free state. In this study, a similar trend was observed, but the calcium value tended to be, and the magnesium value was significantly lower at the onset of neuroleptic-induced EPS than during the mean of an entire pimozide trial.

Practical application of guinea pig telemetry system for QT evaluation.

The purpose of this study was to evaluate a telemetry system for examining QT evaluation in the conscious free-moving guinea pig using 10 reference compounds whose effects on human QT interval are well established: 8 positive references (bepridil, terfenadine, cisapride, haloperidol, pimozide, quinidine, E-4031 and thioridazine), and 2 negative references (propranolol and nifedipine). Pharmacokinetic experiments were also performed for the 8 positive references. Telemetry transmitters were implanted subcutaneously in male Hartley guinea pigs, and the RR and QT intervals were measured. All 8 positive references prolonged QTc (QTc = k x QT/RR(1/2)) 10% or more during the 60 min observation period. When the values of the QTc changes were plotted against the serum concentrations, the resulting curves exhibited an anticlockwise hysteresis loop for all 8 references. In guinea pigs treated with haloperidol, changes of the T-wave shape from positive to flat were observed. The 2 negative references did not prolong the QTc. These findings suggest that the present telemetry guinea pig model is useful for QT evaluation in the early stages of drug development, because of the small body size of guinea pigs and their action potential configuration, which is similar to that of humans.

Prophylactic effects of neuroleptics in symptom-free schizophrenics: roles of dopaminergic and noradrenergic blockers.

A clinical trial was undertaken to determine the role of dopaminergic and noradrenergic blockers in the maintenance treatment of remitted schizophrenics. One hundred and six remitted schizophrenic outpatients were treated with one of nine treatments, viz., thioridazine 25 mg or 75 mg, pimozide 2 mg or 6 mg, and their respective combinations, for 1 year in a double-blind controlled study employing a randomized design. The data from a previous study were utilized as a retrospective placebo group. Pimozide prolonged the number of symptom-free days in a dose-dependent manner and did so more markedly than thioridazine. Combined administration of pimozide and thioridazine prolonged the number of symptom-free days to a greater extent than their single administration. However, an inverted U-shaped dose-response curve was obtained with the combined administration of these agents. These data suggest that both the dopaminergic and noradrenergic blocking action of neuroleptics are important in preventing relapse in remitted schizophrenics.

Prediction of early relapse after pimozide discontinuation by response to d-amphetamine during pimozide treatment.

Thirteen schizophrenic patients underwent a d-amphetamine infusion while being treated with pimozide, an antipsychotic agent and a relatively specific dopamine (DA) blocker. Six patients who showed an acute increase in psychosis with d-amphetamine relapsed within 3 months after pimozide withdrawal. Of the seven patients who did not change in psychosis with d-amphetamine, six did not relapse after pimozide discontinuation (Fisher's exact test, p less than 0.005). Retrospectively, the psychosis-inducing effects of d-amphetamine were found to be predictive of psychotic exacerbation following subsequent pimozide withdrawal. Pimozide failed to block the psychotogenic effects of d-amphetamine in six patients indicating that other mechanisms besides DA may play a role in psychotic decompensation. The d-amphetamine infusion paradigm should be studied further to determine its clinical value for identifying which schizophrenic patients are at risk for relapse after discontinuation of neuroleptic treatment.

Controlled study of erythrocyte choline in Tourette syndrome.

Erythrocyte and plasma choline parameters were compared in children (n = 63), aged 6-18 years, suffering from Tourette Syndrome (TS), their parents (n = 57), their unaffected siblings (n = 38), and an adult control group (n = 57). Factors such as severity of illness, medication status, and gender had no effect on erythrocyte choline. TS patients showed elevations in erythrocyte choline level compared to controls. Furthermore, the erythrocyte choline concentration in TS patients with a history of TS or chronic motor tic disorder (CMT) in first-degree relatives showed a positive correlation with that of their parents (r2 = 0.6, p less than 0.03). Erythrocyte choline values in TS patients without such positive family history do not demonstrate a familial relationship. Positive history of TS or CMT in first-degree relatives accounts for the observation of elevated erythrocyte choline in unaffected siblings of TS patients. Age effects on erythrocyte choline were found in the TS patients only (r = -0.14, p less than 0.04) and not in parents, siblings, or normal controls. A gender effect on plasma choline was noted with male levels 23% higher than in females. The present findings support the utility of erythrocyte choline as a marker for the familial expression of the TS diathesis.

Effect of clarithromycin on the pharmacokinetics and pharmacodynamics of pimozide in healthy poor and extensive metabolizers of cytochrome P450 2D6 (CYP2D6).

BACKGROUND: The use of pimozide is associated with prolongation of the QT interval and fatal ventricular arrhythmia. We recently reported 2 fatal cases in patients taking pimozide and clarithromycin and we have shown that clarithromycin inhibits CYP3A-mediated metabolism of pimozide in vitro. In this study, we examined the effect of clarithromycin on pimozide pharmacokinetics and QT interval changes in a total of 12 healthy subjects (7 men and 5 women), documented as extensive metabolizers or poor metabolizers of CYP2D6. METHODS: In a randomized, double-blind placebo-controlled crossover design, subjects were given a single 6-mg oral dose of pimozide after 5 days of treatment with clarithromycin (500 mg twice a day) or a placebo pill. Blood samples were obtained before and for 96 hours after pimozide administration, and plasma pimozide and clarithromycin concentrations were measured by HPLC. Electrocardiograms for the analysis of the QTc intervals were recorded immediately before each blood sample. RESULTS: Pimozide significantly lengthened QTc interval in the first 20 hours in both the placebo-treated groups (delta QTcmax = 13.3 +/- 5.3 ms; P = .003) and clarithromycin-treated groups (delta QTcmax = 15.7 +/- 9.5 ms; P = .005) compared with baseline values. This is consistent with an effect of the parent drug. Clarithromycin caused a significant increase in the peak plasma concentration (P = .015), terminal elimination half-life (P = .003), and area under the plasma concentration-time curve (P = .024) and a decrease in the clearance (P = .029) of pimozide. Mean QTcmax observed within 20 hours of pimozide administration was significantly greater in the clarithromycin-treated group (23.8 +/- 12.2 ms; P = .0397) than in the placebo-treated group (16.8 +/- 6 ms). There was no significant effect of CYP2D6 or gender on the pharmacokinetics or pharmacodynamics of pimozide. CONCLUSIONS: A single 6-mg oral dose of pimozide resulted in measurable QT interval changes. Clarithromycin inhibited CYP3A-mediated pimozide metabolism and the resulting elevation in plasma concentrations may increase the risk of pimozide cardiotoxicity.

Dissociation of the attentional and motivational effects of pimozide on the threshold for rewarding brain stimulation.

The decreased sensitivity of animals to rewarding brain stimulation caused by pimozide has been interpreted as a selective pharmacologic blockade of central reward pathways rather than a nonspecific disruption of performance. In an attempt to confirm this hypothesis, the effects of pimozide on both reward and detection thresholds for intracranial stimulation delivered to the medial forebrain bundle-lateral hypothalamic area (MFB-LH) were determined in four animals. The drug caused a systematic increase in the reward threshold of each subject but had no such effect on the detection threshold. We conclude that pimozide selectively inhibits the rewarding effects of brain stimulation, and that therefore, the D2 dopamine receptor has a major role in activating central reward pathways subserving pharmacologic and electrical reinforcement. The dual anhedonic/antipsychotic effects of neuroleptic medication are discussed as a possible paradox of central importance to the psychopathology of schizophrenia.

Pimozide: a review of its pharmacological properties and therapeutic uses in psychiatry.

Pimozide 1-(1-[4,4-bis(4-fluorophenyl)butyl]-4-peperidinyl)-2-benzimidazolone, is the first of a new series of psychotropic drugs, the kiphenylbutylpiperidines. It is advocated for once-daily use as maintenance therapy in chronic schizophrenia and for the treatment of psychic and functional disorders induced by personality traits. Published data suggest that in chronic schizophrenia, pimozide 4 to 6mg daily is indistinguishable from maintenance doses of chlorpromazine, fluphenazine, flupenthixol, perphenazine, or thioidazine. Patient groups have usually been to small to allow statistically significant differences to be apparent, but in some trials pimozide was significantly superior to trifluoperzine and to haloperidol. On present evidence, pimozide has no place in the hyperactive, aggressive type of patient or in treating the acute phase of schizophrenia, probably because of its relative lack of sedative properties compared with many antipsychotic drugs. The incidence and severity of extrapyramidal reactions with pimozide are low, but suitably designed controlled studies are needed to determine whether its use leads to a reduction in the requirement for antiparkinsonian medication. In anxious patients, pimozide seems to offer no advantages over currently available anxiolytic agents, either in terms of efficacy or incidence of side-effects. Claims for a specific effect against anxiety associated with psychosis or disturbed personality traits remain unproven.

Low-dose pimozide augmentation of serotonin reuptake blockers in the treatment of trichotillomania.

BACKGROUND: There is some overlap in the phenomenology and treatment response of trichotillomania, Gilles de la Tourette's syndrome (TS), and obsessive compulsive disorder (OCD). Neuroleptics may play a role in the treatment of TS as well as refractory OCD, and the question arises whether this also holds for trichotillomania. METHOD: Patients with trichotillomania were treated in open clinical practice with serotonin reuptake blockers. When symptom resolution was incomplete or symptom relapse occurred, low-dose pimozide was added. RESULTS: In six out of seven patients, the addition of pimozide to a serotonin reuptake blocker led to an improvement in hair pulling. In patients who were able to tolerate their medication, this response was sustained. CONCLUSION: Augmentation of serotonergic agents with dopamine blockers may play a role in the treatment of trichotillomania. Further controlled trials of pimozide augmentation in trichotillomania are needed.

Effects of pimozide on the acquisition, maintenance, and extinction of an amphetamine-induced taste aversion.

Different groups of rats were pretreated with the dopamine receptor blocker, pimozide (0.25, 0.5, or 1.0 mg/kg), in an attempt to investigate the role of dopaminergic transmission in the acquisition, maintenance, and extinction of a taste aversion produced by d-amphetamine dulphate (1.0 or 2.0 mg/kg). In the first phase of the experiment, all doses of pimozide attenuated but did not block the acquisition of the aversion produced by 1.0 mg/kg but not by 2.0 mg/kg amphetamine. In Phase II, pimozide pretreatment was suspended to allow the attenuated groups to acquire the aversion and then reintroduced in Phase III. In this phase all groups continued to avoid the taste, indicating a failure of pimozide to affect the maintenance of the avoidance response. When amphetamine treatment was suspended in Phase IV, pimozide accelerated the extinction, especially in those groups that had previously received the 1.0 mg/kg dose of amphetamine. These results are discussed with reference to dopaminergic mechanisms in avoidance learning and a pimozide-mediated reduction in the functional strength of amphetamine as an unconditioned stimulus.